Enzymatic production of alpha-1,3-glucan

ABSTRACT

A method for producing insoluble alpha-1,3-glucan is disclosed. Embodiments of the method comprise providing (i) oligosaccharides that comprise alpha-1,3 and alpha-1,6 glycosidic linkages, or (ii) oligosaccharides derived from a glucosyltransferase reaction; and contacting at least water, sucrose, a glucosyltransferase enzyme, and the oligosaccharides provided in the first step. Glucosyltransferase reaction compositions embodying such a method, and insoluble products thereof, are also disclosed. Yield and other product benefits can be realized when practicing the disclosed subject matter.

This application is a continuation of U.S. application Ser. No.15/985,831 (filed May 22, 2018, now U.S. Pat. No. 10/774,352), whichclaims the benefit of U.S. Provisional Application Nos. 62/509,915(filed May 23, 2017) and 62/519,217 (filed Jun. 14, 2017), all of whichprior applications are incorporated herein by reference in theirentirety.

FIELD

This present disclosure is in the field of enzymatic processes. Forexample, the disclosure pertains to glucosyltransferase reactionscomprising added oligosaccharides.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named

-   20180522_CL6007USNP_SequenceListing created on May 18, 2018, and    having a size of about 157 kilobytes and is filed concurrently with    the specification. The sequence listing contained in this    ASCII-formatted document is part of the specification and is herein    incorporated by reference in its entirety.

BACKGROUND

Driven by a desire to use polysaccharides in various applications,researchers have explored for polysaccharides that are biodegradable andthat can be made economically from renewably sourced feedstocks. Onesuch polysaccharide is alpha-1,3-glucan, an insoluble glucan polymercharacterized by having alpha-1,3-glycosidic linkages. This polymer hasbeen prepared, for example, using a glucosyltransferase enzyme isolatedfrom Streptococcus salivarius (Simpson et al., Microbiology141:1451-1460, 1995). Also for example, U.S. Pat. No. 7,000,000disclosed the preparation of a spun fiber from enzymatically producedpoly alpha-1,3-glucan.

Enzymatic synthesis of various glucan polymers has been performed inreactions in which polysaccharides (e.g., dextran) or oligosaccharides(e.g., from hydrolyzed polysaccharide) have been added to affectglucosyltransferase function (e.g., Koga et al., 1983, J. Gen.Microbiol. 129:751-754; Komatsu et al., 2011, FEBS J. 278:531-540;Simpson et al.; O'Brien et al., U.S. Pat. No. 8,642,757). Despite thesedisclosures, there is little understanding with regard to modulatingglucosyltransferase reactions for insoluble alpha-1,3-glucan synthesis.

SUMMARY

In one embodiment, the present disclosure concerns a method forproducing insoluble alpha-1,3-glucan comprising:

-   -   (a) providing oligosaccharides that:        -   comprise alpha-1,3 and alpha-1,6 glycosidic linkages, and/or        -   (ii) are produced from a glucosyltransferase reaction;    -   (b) contacting at least water, sucrose, the oligosaccharides,        and a glucosyltransferase enzyme that synthesizes insoluble        alpha-1,3-glucan, wherein insoluble alpha-1,3-glucan is        produced; and    -   (c) optionally, isolating the insoluble alpha-1,3-glucan        produced in step (b).

In another embodiment, the present disclosure concerns a reactioncomposition for producing insoluble alpha-1,3-glucan, the reactioncomposition comprising at least water, sucrose, a glucosyltransferaseenzyme that synthesizes insoluble alpha-1,3-glucan, andoligosaccharides, wherein the oligosaccharides are added duringpreparation of the reaction composition and (i) comprise alpha-1,3 andalpha-1,6 glycosidic linkages, and/or (ii) are produced from aglucosyltransferase reaction, wherein insoluble alpha-1,3-glucan isproduced in the reaction composition.

In another embodiment, the present disclosure concerns a compositioncomprising insoluble alpha-1,3-glucan produced according to any methodherein of producing insoluble alpha-1,3-glucan.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

FIG. 1 : ¹H-NMR spectra of a concentrated oligosaccharide preparation(see Example 2).

FIG. 2 : The graph shows the aqueous slurry viscosity of eachalpha-1,3-glucan product made in three successive reactions, where thesecond and third reactions incorporated filtrate derived from the firstand second reactions, respectively (see Example 10). Squares, circlesand diamonds indicate, respectively, viscosity measurements taken withaqueous slurries of alpha-1,3-glucan produced in first, second and thirdreactions. Shear rate units are in 1/s (shown as “s−1”).

TABLE 1 Summary of Nucleic Acid and Protein Sequence IdentificationNumbers Nucleic acid Protein Description SEQ ID NO. SEQ ID NO. GTFJ orGTF 7527, Streptococcus salivarius. The 1 ^(a) 2 first 42 amino acids ofthe protein are deleted (1477 aa) compared to GENBANK Identification No.47527; a start methionine is included. GTF 0874, Streptococcus sobrinus.The first 156 3 ^(a) 4 amino acids of the protein are deleted comparedto (1435 aa) GENBANK Identification No. 450874; a start methionine isincluded. GTF 1724, Streptococcus downei. The first 162 5 ^(a) 6 aminoacids of the protein are deleted compared to (1436 aa) GENBANKIdentification No. 121724; a start methionine is included. GTF 1724-T1,Streptococcus downei. The first 217 7 amino acids and the last 530 aminoacids of the (851 aa) protein are deleted compared to GENBANKIdentification No. 121724; a start methionine is included. GTFJ-T1 orGTF 7527-T1, Streptococcus salivarius. 8 The first 230 amino acids andthe last 384 amino (905 aa) acids of the protein are deleted compared toGENBANK Identification No. 47527; a start methionine is included. GTF6855, Streptococcus salivarius SK126. The first 9 178 amino acids of theprotein are deleted compared (1341 aa) to GENBANK Identification No.228476855; a start methionine is included. GTF 5926, Streptococcusdentirousetti. The first 144 10 amino acids of the protein are deletedcompared to (1323 aa) GENBANK Identification No. 167735926; a startmethionine is included. GTF 2765, unknown Streptococcus sp. C150. The 11first 193 amino acids of the protein are deleted (1340 aa) compared toGENBANK Identification No. 322372765; a start methionine is included.GTF 0427, Streptococcus sobrinus. The first 156 12 amino acids of theprotein are deleted compared to (1435 aa) GENBANK Identification No.940427; a start methionine is included. GTF 2919, Streptococcussalivarius PS4. The first 92 13 amino acids of the protein are deletedcompared to (1340 aa) GENBANK Identification No. 383282919; a startmethionine is included. GTF 2678, Streptococcus salivarius K12. Thefirst 14 188 amino acids of the protein are deleted compared (1341 aa)to GENBANK Identification No. 400182678; a start methionine is included.GTF 3929, Streptococcus salivarius JIM8777. The 15 first 178 amino acidsof the protein are deleted (1341 aa) compared to GENBANK IdentificationNo. 387783929; a start methionine is included. “GTF 7527-short” (shorterversion of GTFJ), 16 Streptococcus salivarius. The first 178 amino acidsof (1341 aa) the protein are deleted compared to GENBANK IdentificationNo. 47527; a start methionine is included. ^(a) This DNA coding sequenceis codon-optimized for expression in E. coli and is merely disclosed asan example of a suitable coding sequence.

DETAILED DESCRIPTION

The disclosures of all cited patent and non-patent literature areincorporated herein by reference in their entirety.

Unless otherwise disclosed, the terms “a” and “an” as used herein areintended to encompass one or more (i.e., at least one) of a referencedfeature.

Where present, all ranges are inclusive and combinable, except asotherwise noted. For example, when a range of “1 to 5” is recited, therecited range should be construed as including ranges “1 to 4”, “1 to3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

The term “saccharide” as used herein refers to monosaccharides and/ordisaccharides/oligosaccharides, unless otherwise noted. A “disaccharide”herein refers to a carbohydrate having two monosaccharides joined by aglycosidic linkage. An “oligosaccharide” herein can refer to acarbohydrate having 2 to 15 monosaccharides, for example, joined byglycosidic linkages. An oligosaccharide can also be referred to as an“oligomer”. Monosaccharides (e.g., glucose and/or fructose) comprisedwithin disaccharides/oligosaccharides can be referred to as “monomerunits”, “monosaccharide units”, or other like terms.

The terms “alpha-glucan”, “alpha-glucan polymer” and the like are usedinterchangeably herein. An alpha-glucan is a polymer comprising glucosemonomeric units linked together by alpha-glycosidic linkages. In typicalembodiments, an alpha-glucan herein comprises at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages. Alpha-1,3-glucanis an example of an alpha-glucan.

The terms “alpha-1,3-glucan”, “poly alpha-1,3-glucan”, “alpha-1,3-glucanpolymer” and the like are used interchangeably herein. Alpha-1,3-glucanis a polymer comprising glucose monomeric units linked together byglycosidic linkages (i.e., glucosidic linkages), typically wherein atleast about 50% of the glycosidic linkages are alpha-1,3-glycosidiclinkages. Alpha-1,3-glucan in certain embodiments comprises at leastabout 90% or 95% alpha-1,3 glycosidic linkages. Most or all of the otherlinkages in alpha-1,3-glucan herein typically are alpha-1,6, though somelinkages may also be alpha-1,2 and/or alpha-1,4.

The terms “glycosidic linkage”, “linkage”, “glycosidic bond” and thelike are used interchangeably herein and refer to the type of covalentbond that joins a carbohydrate (sugar) molecule to another carbohydratemolecule. All glycosidic linkages disclosed herein are alpha-glucosidiclinkages, except as otherwise noted. A “glucosidic linkage” refers to aglycosidic linkage between an alpha-D-glucose and another carbohydratemolecule. “Alpha-D-glucose” herein is also be referred to as “glucose”.The terms “alpha-1,3 glucosyl-glucose linkage”, “alpha-1,3glucose-glucose linkage” and “glucose-alpha 1,3-glucose” herein refer toan alpha-1,3 glycosidic linkage. The terms “alpha-1,6 glucosyl-glucoselinkage”, “alpha-1,6 glucose-glucose linkage” and “glucose-alpha1,6-glucose” herein refer to an alpha-1,6 glycosidic linkage.

The glycosidic linkage profile of any polysaccharide herein (e.g.,alpha-1,3-glucan, oligosaccharides) can be determined using any methodknown in the art. For example, a linkage profile can be determined usingmethods using nuclear magnetic resonance (NMR) spectroscopy (e.g., ¹³CNMR or ¹H NMR). These and other methods that can be used are disclosedin, for example, Food Carbohydrates: Chemistry, Physical Properties, andApplications (S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysisof Polysaccharides, Taylor & Francis Group LLC, Boca Raton, Fla., 2005),which is incorporated herein by reference.

The “molecular weight” of large alpha-glucan polymers herein can berepresented as weight-average molecular weight (Mw) or number-averagemolecular weight (Mn), the units of which are in Daltons or grams/mole.Alternatively, the molecular weight of large alpha-glucan polymers canbe represented as DPw (weight average degree of polymerization) or DPn(number average degree of polymerization). The molecular weight ofsmaller alpha-glucan polymers such as oligosaccharides typically can beprovided as “DP” (degree of polymerization), if desired, which simplyrefers to the number of glucoses comprised within the alpha-glucan.Various means are known in the art for calculating these variousmolecular weight measurements such as with high-pressure liquidchromatography (HPLC), size exclusion chromatography (SEC), or gelpermeation chromatography (GPC).

The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”,“glucansucrase” and the like are used interchangeably herein. Theactivity of a glucosyltransferase herein catalyzes the reaction of thesubstrate sucrose to make the products alpha-glucan and fructose. Otherproducts (by-products) of a glucosyltransferase reaction can includeglucose, various soluble gluco-oligosaccharides, and leucrose. Wild typeforms of glucosyltransferase enzymes generally contain (in theN-terminal to C-terminal direction) a signal peptide (which is typicallyremoved by cleavage processes), a variable domain, a catalytic domain,and a glucan-binding domain. A glucosyltransferase herein is classifiedunder the glycoside hydrolase family 70 (GH70) according to the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009).

The term “glucosyltransferase catalytic domain” herein refers to thedomain of a glucosyltransferase enzyme that providesalpha-glucan-synthesizing activity to a glucosyltransferase enzyme. Aglucosyltransferase catalytic domain typically does not require thepresence of any other domains to have this activity.

The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucansynthesis reaction”, “reaction composition”, “reaction formulation” andthe like are used interchangeably herein and generally refer to areaction that initially comprises water, sucrose, at least one activeglucosyltransferase enzyme, and optionally other components. Componentsthat can be further present in a glucosyltransferase reaction typicallyafter it has commenced include fructose, glucose, leucrose, solublegluco-oligosaccharides (e.g., DP2-DP7) (such may be considered asproducts or by-products, depending on the glucosyltransferase used),and/or insoluble alpha-glucan product(s) of DP8 or higher (e.g., DP100and higher). It would be understood that certain glucan products, suchas alpha-1,3-glucan with a degree of polymerization (DP) of at least 8or 9, are water-insoluble (“insoluble alpha-1,3-glucan) and thus notdissolved in a glucan synthesis reaction, but rather may be present outof solution (e.g., by virtue of having precipitated from the reaction).It is in a glucan synthesis reaction where the step of contacting water,sucrose and a glucosyltransferase enzyme is performed. The term “undersuitable reaction conditions” as used herein refers to reactionconditions that support conversion of sucrose to alpha-glucan product(s)via glucosyltransferase enzyme activity.

A “control” reaction as used herein can refer to a glucosyltransferasereaction to which no oligosaccharides comprising (collectivelycomprising) alpha-1,3 and alpha-1,6 glycosidic linkages have beendirectly added to the reaction. All the other features (e.g., sucroseconcentration, temperature, pH, type of GTF) of a control reactionsolution can be the same as the reaction composition to which it isbeing compared.

The “percent dry solids” (percent DS) of a solution herein (e.g.,soluble fraction, aqueous composition) refers to the wt % of all thematerials (i.e., the solids) dissolved in the solution. For example, a100 g solution with 10 wt % DS comprises 10 g of dissolved material.

The “yield” of alpha-1,3-glucan by a glucosyltransferase reaction incertain embodiments represents the weight of alpha-1,3-glucan productexpressed as a percentage of the weight of sucrose substrate that isconverted in the reaction. For example, if 100 g of sucrose in areaction solution is converted to products, and 10 g of the products isalpha-1,3-glucan, the yield of the alpha-1,3-glucan would be 10%. The“yield” of alpha-1,3-glucan by a glucosyltransferase reaction in someaspects represents the molar yield based on the converted sucrose. Themolar yield of an alpha-glucan product can be calculated based on themoles of the alpha-glucan product divided by the moles of the sucroseconverted. Moles of converted sucrose can be calculated as follows:(mass of initial sucrose−mass of final sucrose)/molecular weight ofsucrose [342 g/mol]. These yield calculations (yield based on weight ormoles) can be considered as measures of selectivity of the reactiontoward alpha-1,3-glucan. In some aspects, the “yield” of an alpha-glucanproduct in a glucosyltransferase reaction can be based on the glucosylcomponent of the reaction. Such a yield (yield based on glucosyl) can bemeasured using the following formula:Alpha-Glucan Yield=((IS/2−(FS/2+LE/2+GL+SO))/(IS/2−FS/2))×100%.The fructose balance of a glucosyltransferase reaction can be measuredto ensure that HPLC data, if applicable, are not out of range (90-110%is considered acceptable). Fructose balance can be measured using thefollowing formula:Fructose Balance=((180/342×(FS+LE)+FR)/(180/342×IS))×100%.

In the above two formulae, IS is [Initial Sucrose], FS is [FinalSucrose], LE is [Leucrose], GL is [Glucose], SO is [Soluble Oligomers](gluco-oligosaccharides), and FR is [Fructose] (all concentrations inunits of grams/L and as measured by HPLC, for example).

The term “relative reaction rate” as used herein refers to the rate of aparticular glucan synthesis reaction as compared to another glucansynthesis reaction. For example, if reaction A has a rate of x, andreaction B has a rate of y, then the relative reaction rate of reactionA with respect to the reaction rate of reaction B can be expressed asx/y (x divided by y). The terms “reaction rate” and “rate of reaction”are used interchangeably herein to refer to the change inconcentration/amount of reactant(s) or the change inconcentration/amount of product(s) per unit time per unit of enzyme. AsGTF enzymes are known to follow Michaelis-Menten kinetics, these ratesare typically measured at the beginning of polymerization when theamount of sucrose is well above the Km for the enzyme. In this case, therate is typically measured when the amount of sucrose in the reaction isabove at least about 50 g/L sucrose. Preferred reactant and productherein of a glucan synthesis reaction are, respectively, sucrose andalpha-1,3-glucan.

A “soluble fraction” or “soluble portion” of a glucosyltransferasereaction herein refers to a liquid solution portion of theglucosyltransferase reaction. A soluble fraction can be a portion of, orall of, the liquid solution from a glucosyltransferase reaction, andtypically has been separated from an insoluble glucan productsynthesized in the reaction. A soluble fraction can alternatively bereferred to as a “mother liquor”. An example of a soluble fraction is afiltrate of a glucosyltransferase reaction. Since a soluble fraction cancontain dissolved sugars such as sucrose, fructose, glucose, leucrose,soluble gluco-oligosaccharides, a fraction can also be referred to as a“mixed sugar solution” derived from a glucosyltransferase reaction. Asoluble fraction herein can remain unprocessed following itsacquisition, or alternatively, it can be subjected to one or moreprocessing steps as disclosed herein.

The terms “filtrate”, “glucan reaction filtrate”, and the like are usedinterchangeably herein and refer to a soluble fraction that has beenfiltered away from an insoluble glucan product synthesized in aglucosyltransferase reaction.

The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” andthe like are used interchangeably herein. The percent by volume of asolute in a solution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)”,“weight-weight percentage (% w/w)” and the like are used interchangeablyherein. Percent by weight refers to the percentage of a material on amass basis as it is comprised in a composition, mixture, or solution.

The term “aqueous conditions” and like terms herein refer to a solutionor mixture in which the solvent is at least about 60 wt % water, forexample. A glucosyltransferase reaction herein is performed underaqueous conditions.

A glucan that is “insoluble”, “aqueous-insoluble”, “water-insoluble”(and like terms) (e.g., insoluble alpha-1,3-glucan) does not dissolve(or does not appreciably dissolve) in water or other aqueous conditions,optionally where the aqueous conditions are further characterized tohave a pH of 4-9 (e.g., 6-8) and/or temperature of about 1 to 85° C.(e.g., 20-25° C.). In contrast, glucans such as certain oligosaccharidesherein that are “soluble”, “aqueous-soluble”, “water-soluble” and thelike appreciably dissolve under these conditions.

An “aqueous composition” herein has a liquid component that comprises atleast about 10 wt % water, for example (e.g., liquid component can be atleast about 70%, 80%, 90%, 95% water, or 100% water). Examples ofaqueous compositions include mixtures, solutions, dispersions (e.g.,colloidal dispersions), suspensions and emulsions, for example. Aqueouscompositions in certain embodiments comprise alpha-1,3-glucan asproduced herein that is mixed (e.g., by homogenization) or dissolved(e.g., via dissolution under caustic aqueous conditions such as at a pHof at least 11.0 [as provided using an alkaline solute such NaOH or KOH,for example]) in the aqueous composition. A “non-aqueous composition”herein can be “dry” (e.g., comprises no more than 2.0, 1.5, 1.0, 0.5,0.25, 0.10, 0.05, or 0.01 wt % water) and/or comprise a non-aqueousliquid component (e.g., an organic liquid that can dissolvealpha-1,3-glucan such as N,N-dimethylacetamide (DMAc)/0.5%-5% LiCl).

The term “purified” herein can characterize an oligosaccharidepreparation comprising no more than 25% (dry weight basis) ofsaccharides and/or other non-salt/non-buffer material not embraced bythe above definition of oligosaccharides. As the definition implies, apurified oligosaccharide preparation can optionally comprise saltsand/or buffers, the level of neither of which are determinative ofoligosaccharide purity. The term “unpurified” herein can characterize anoligosaccharide preparation comprising more than 25% (dry weight basis)saccharides, and/or other non-salt/non-buffer material, not embraced bythe above definition of oligosaccharides.

The term “viscosity” as used herein refers to the measure of the extentto which a fluid (aqueous or non-aqueous) resists a force tending tocause it to flow. Various units of viscosity that can be used hereininclude centipoise (cP, cps) and Pascal-second (Pa·s), for example. Acentipoise is one one-hundredth of a poise; one poise is equal to 0.100kg·m⁻¹·s⁻¹. Viscosity can be reported as “intrinsic viscosity” (IV, η,units of mL/g) in some aspects; this term refers to a measure of thecontribution of a glucan polymer to the viscosity of a liquid (e.g.,solution) comprising the glucan polymer.

The term “increased” as used herein can refer to a quantity or activitythat is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% morethan the quantity or activity for which the increased quantity oractivity is being compared. The terms “increased”, “elevated”,“enhanced”, “greater than”, “improved” and the like are usedinterchangeably herein. These terms can be used to characterize the“over-expression” or “up-regulation” of a polynucleotide encoding aprotein, for example.

The terms “sequence identity”, “identity” and the like as used hereinwith respect to polynucleotide or polypeptide sequences herein refer tothe nucleic acid residues or amino acid residues in two sequences thatare the same when aligned for maximum correspondence over a specifiedcomparison window. Thus, “percentage of sequence identity”, “percentidentity” and the like refer to the value determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the results by 100 to yield the percentage of sequenceidentity. It would be understood that, when calculating sequenceidentity between a DNA sequence and an RNA sequence, T residues of theDNA sequence align with, and can be considered “identical” with, Uresidues of the RNA sequence. For purposes of determining “percentcomplementarity” of first and second polynucleotides, one can obtainthis by determining (i) the percent identity between the firstpolynucleotide and the complement sequence of the second polynucleotide(or vice versa), for example, and/or (ii) the percentage of basesbetween the first and second polynucleotides that would create canonicalWatson and Crick base pairs.

Percent identity can be readily determined by any known method,including but not limited to those described in: 1) ComputationalMolecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991), all of which are incorporated herein byreference.

Preferred methods for determining percent identity are designed to givethe best match between the sequences tested. Methods of determiningidentity and similarity are codified in publicly available computerprograms, for example. Sequence alignments and percent identitycalculations can be performed using the MEGALIGN program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.),for example. Multiple alignment of sequences can be performed, forexample, using the Clustal method of alignment which encompasses severalvarieties of the algorithm including the Clustal V method of alignment(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values cancorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method can be KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, theseparameters can be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4. Additionally, the Clustal W method of alignment can be used(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J. D. et al,Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). Default parameters for multiple alignment(protein/nucleic acid) can be: GAP PENALTY=10/15, GAP LENGTHPENALTY=0.2/6.66, Delay Divergen Seqs (%)=30/30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

Various polypeptide amino acid sequences and polynucleotide sequencesare disclosed herein as features of certain embodiments. Variants ofthese sequences that are at least about 70-85%, 85-90%, or 90%-95%identical to the sequences disclosed herein can be used or referenced.Alternatively, a variant amino acid sequence or polynucleotide sequencecan have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. Thevariant amino acid sequence or polynucleotide sequence has the samefunction/activity of the disclosed sequence, or at least about 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of the function/activity of the disclosedsequence. Any polypeptide amino acid sequence disclosed herein notbeginning with a methionine can typically further comprise at least astart-methionine at the N-terminus of the amino acid sequence. Incontrast, any polypeptide amino acid sequence disclosed herein beginningwith a methionine can optionally lack such a methionine residue.

It is believed that the compositions (e.g., insoluble alpha-1,3-glucanin certain embodiments) and glucosyltransferase reactions/methodsdisclosed herein are synthetic and non-naturally occurring. Thus, suchaspects herein can optionally be characterized as being “isolated”,which means for example that they can be carried out in a manner thatdoes not occur in nature. It is further believed that theproperties/effects of the aforementioned subject matter are notnaturally occurring.

It is now disclosed that yield and other product benefits can berealized when applying certain oligosaccharides to glucosyltransferasereactions for insoluble alpha-1,3-glucan production.

Embodiments of the present disclosure concern a method for producinginsoluble alpha-1,3-glucan. The method comprises:

-   -   (a) providing oligosaccharides that:        -   (i) comprise alpha-1,3 and alpha-1,6 glycosidic linkages,            and/or        -   (ii) are produced from a glucosyltransferase reaction; and    -   (b) contacting at least water, sucrose, a glucosyltransferase        enzyme, and the oligosaccharides provided in step (a). Insoluble        alpha-1,3-glucan is produced in this method. In certain        embodiments, the yield of insoluble alpha-1,3-glucan product is        increased compared to the yield of insoluble alpha-1,3-glucan        that would have been produced if step (b) was performed without        the oligosaccharides provided in step (a). Insoluble        alpha-1,3-glucan produced in step (b) of the method can        optionally be isolated.

Significantly, oligosaccharides comprising alpha-1,3 and alpha-1,6glycosidic linkages are disclosed herein to modulate the activity ofglucosyltransferase enzymes that produce insoluble alpha-1,3-glucan.Such oligosaccharides can optionally be derived as a by-product of aglucosyltransferase reaction as disclosed herein. Thus, the disclosedmethod in certain embodiments represents an advantageous way to recycleoligosaccharide by-products of a glucosyltransferase reaction. Also ofsignificance herein is that oligosaccharide by-products are useful formodulating glucosyltransferase activity even when provided in anunpurified state such as in a filtrate obtained from aglucosyltransferase reaction.

Oligosaccharides in certain embodiments of the present disclosurecomprise alpha-1,3 glycosidic linkages and alpha-1,6 glycosidiclinkages. For example, oligosaccharides herein can comprise about60-99%, 60-95%, 70-90%, or 80-90% alpha-1,3 glycosidic linkages, andabout 1-40%, 5-40%, 10-30%, or 1-10° A alpha-1,6 glycosidic linkages.Still, in some aspects, oligosaccharides can comprise about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or range betweenany two of these values) alpha-1,3 glycosidic linkages, and about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% (or range between any twoof these values) alpha-1,6 glycosidic linkages. Such a linkage profilecan characterize oligosaccharides of any molecular weight herein (e.g.,DP2-7, DP2-8, DP2-9, or DP2-10). The aforementioned linkage profiles canoptionally characterize gluco-oligosaccharides.

Oligosaccharides herein can, for instance, “collectively comprise” anyof the foregoing linkage profiles. By “collectively comprise”, it ismeant that the linkage profile of a mixture of various oligosaccharidesis based on the combination of all the linkages present in the mixture.Oligosaccharides useful herein can therefore comprise particularoligosaccharide species containing only alpha-1,3 glycosidic linkages,only alpha-1,6 glycosidic linkages, and/or both alpha-1,3 and alpha-1,6glycosidic linkages, just so long that the total linkage profile of allthe oligosaccharide species present falls under any of the foregoinglinkage profiles (e.g., ˜78% alpha-1,3 linkages and ˜22% alpha-1,6linkages, or ˜87-88% alpha-1,3 linkages and ˜7% alpha-1,6 linkages).Oligosaccharides in certain aspects do not comprise/collectivelycomprise 100% alpha-1,3 glycosidic linkages or 100% alpha-1,6 glycosidiclinkages.

Gluco-oligosaccharides herein preferably contain mostly alpha-1,3 andalpha-1,6 glycosidic linkages. For example, at least about 95%, 96%,97%, 98%, 99%, or 100% of the total linkages of the oligosaccharides arealpha-1,3 and alpha-1,6 glycosidic linkages. Other linkages, if presentin the oligosaccharides, may be alpha-1,4 (e.g., ≤1.5% or 1%) oralpha-1,2 (e.g., 1% or 0.7%) glycosidic linkages, for example.

Oligosaccharides herein can have a degree of polymerization (DP) of 2 to15 in some aspects. As examples, the oligosaccharides can have a DP of2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, or 2-15. As would beunderstood in the art, a group of oligosaccharides herein can bereferenced with respect to a DP number or range, which specifies thenumber or range of monomeric units in individual oligosaccharide speciesin the group. For example, DP2-7 oligosaccharides typically comprise amixture of DP2, DP3, DP4, DP5, DP6 and DP7 oligosaccharides. Theaforementioned oligosaccharides can optionally be referred to asgluco-oligosaccharides.

The distribution of oligosaccharides in a composition used to provideoligosaccharides herein can vary. For example, a composition comprisingoligosaccharides of DP 2-7 can comprise oligosaccharides having adistribution profile that is the same or similar to that disclosed belowin Table 5. Thus, a composition comprising DP2-7 oligosaccharides cancomprise, for example, about 5-15 wt % (e.g., ˜9-11 wt %) DP2, about19-29 wt % (e.g., ˜23-25 wt %) DP3, about 27-37 wt % (e.g., ˜31-33 wt %)DP4, about 15-25 wt % (e.g., ˜19-21 wt %) DP5, about 3-13 wt % (e.g.,˜7-9 wt %) DP6, and about 1 to 10 wt % (e.g., ˜4-6 wt %) DP7oligosaccharides on the basis of the saccharide components in thecomposition or on a dry weight basis. In some aspects, a compositioncomprising oligosaccharides of DP 2-7 can comprise oligosaccharideshaving a distribution profile that is the same or similar to thatdisclosed below in Table 16. Thus, a composition comprising DP2-7oligosaccharides can comprise, for example, about 6-16 wt % (e.g.,˜10-12 wt %) DP2, about 18-28 wt % (e.g., ˜22-24 wt %) DP3, about 23-33wt % (e.g., ˜27-29 wt %) DP4, about 16-26 wt % (e.g., ˜20-22 wt %) DP5,about 7-17 wt % (e.g., ˜11-13 wt %) DP6, and about 1 to 10 wt % (e.g.,˜4-6 wt %) DP7 oligosaccharides on the basis of the saccharidecomponents in the composition or on a dry weight basis. The exact DPdistribution is not believed to be critical to the present disclosure;other distributions should provide the same behavior described herein.

In certain embodiments of the present disclosure, the oligosaccharidescan be purified or unpurified. Purified oligosaccharides can be providedusing any suitable means known in the art, such as via chromatography asdisclosed in the below Examples, or by following the disclosure ofEuropean Patent Publ. No. EP2292803B1, which is incorporated herein byreference. Purified oligosaccharides can be provided, for example, in adry form or an aqueous form (aqueous solution), either of which mayoptionally also contain one or more salts (e.g., NaCl) and/or buffers. Apurified oligosaccharide preparation in certain embodiments can compriseless than about 25, 20, 15, 10, 5, 2.5, 2, 1.5, 1.0, 0.5, or 0.1 wt % of(i) saccharides that are not embraced by the definition ofoligosaccharides as disclosed herein (e.g., oligosaccharides herein arenot monosaccharides or DP11+ saccharides) and/or (ii) othernon-salt/non-buffer material.

Unpurified oligosaccharides can be used in certain embodiments of thepresent disclosure. An unpurified oligosaccharide preparation cancomprise, for example, more than about 2, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, or 95% wt % saccharides, and/or other non-salt/non-buffermaterial, not embraced by the definition of oligosaccharides asdisclosed herein. An example of an unpurified oligosaccharidepreparation herein is a soluble fraction (e.g., filtrate) from aglucosyltransferase reaction. Other “non-salt/non-buffer material” thatcan be present in a soluble fraction herein include sucrose, fructose,glucose, leucrose, and glucosyltransferase protein, for example.

Oligosaccharides provided in step (a) of the disclosed method can beproduced from (“derived from”, derivable or obtainable from) aglucosyltransferase reaction. The oligosaccharides can be a by-productof a glucosyltransferase reaction, for example. Such a by-product can befrom a glucosyltransferase reaction that synthesizes insolublealpha-1,3-glucan in certain embodiments.

A glucosyltransferase reaction from which oligosaccharides herein can beproduced generally refers to an aqueous composition comprising at leastsucrose, water and one active glucosyltransferase enzyme, and optionallyother components. Other components that can be in a glucosyltransferasereaction include at least fructose, glucose, leucrose, andgluco-oligosaccharides. It would be understood that certain glucanproducts, such as alpha-1,3-glucan with a DP of at least 8 or 9, can bewater-insoluble and thus are not dissolved in a glucosyltransferasereaction, but rather present out of solution. Thus, oligosaccharidesherein can be derived from a glucosyltransferase reaction that producesan insoluble glucan product (e.g., alpha-1,3-glucan).

A glucosyltransferase reaction from which oligosaccharides may bederived can comprise one or more of the following types ofglucosyltransferase enzymes: a GTF that produces alpha-1,3-glucan withat least 50% alpha-1,3 glycosidic linkages (e.g., GTF's disclosed hereinthat can also be used as a GTF in the disclosed method itself),mutansucrase, dextransucrase, reuteransucrase, alternansucrase. Incertain embodiments, oligosaccharides are from a reaction comprisingonly one or two glucosyltransferases that produce insolublealpha-1,3-glucan.

Oligosaccharides herein are typically derived from a glucosyltransferasereaction at a stage in which by-product oligosaccharides have formed inthe reaction. Oligosaccharides form throughout a polymerizationreaction. For example, oligosaccharides can be from aglucosyltransferase reaction that is only partially complete to nearlycomplete (e.g., 80 to 90% complete) or at completion (e.g. >95%complete), where completion is defined as the amount of sucrose consumeddivided by the total amount of sucrose fed to the polymerization.

Oligosaccharides in certain embodiments of the present disclosure can beprovided as a soluble fraction of a glucosyltransferase reaction. Asoluble fraction herein can be processed or unprocessed. A solublefraction can be a portion of, or all of, the liquid solution from aglucosyltransferase reaction. Typically, a soluble fraction has beenseparated from solid glucan product(s) synthesized in the reaction; thisapplies to glucan products that are insoluble in water such asalpha-1,3-glucan which fall out of solution during their synthesis. Asoluble fraction in certain embodiments of the present disclosure isfrom a glucosyltransferase reaction that produces alpha-1,3-glucan.However, a soluble fraction can optionally be from a glucosyltransferasereaction that does not produce an insoluble glucan product (e.g.,dextran).

The volume of a collected soluble fraction (before optionally processingthe soluble fraction, see below) in certain embodiments can be at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the volumeof the glucosyltransferase reaction from which it is obtained.Typically, in glucosyltransferase reactions producing an insolubleglucan (e.g., alpha-1,3-glucan), the soluble fraction will be a portionof (not all of) the liquid solution component of the reaction. A solublefraction can be obtained at a stage of a glucosyltransferase reaction inwhich by-product oligosaccharides have formed in the reaction. Forexample, a soluble fraction can be from a glucosyltransferase reactionthat is only partially complete to nearly complete (e.g., 80 to 90%complete) or at completion (e.g. >95% complete).

Examples of a soluble fraction of a glucosyltransferase reaction incertain embodiments include filtrates and supernatants. Thus, a solublefraction herein can be obtained (separated) from a glucosyltransferasereaction using a funnel, filter (e.g., a surface filter such as a rotaryvacuum-drum filter, cross-flow filter, screen filter, belt filter, screwpress, or filter press with or with membrane squeeze capability; or adepth filter such as a sand filter), centrifuge, and/or any other methodor equipment known in the art that allows removal of some or all liquidsfrom solids. Filtration can be by gravity, vacuum, or press filtration,for example. Filtration preferably removes all or most insoluble glucan;any filter material (e.g., cloth, metal screen, or filter paper) with anaverage pore size (e.g., ˜10-50 micron) sufficient to remove solids fromliquids can be used. A soluble fraction typically retains all or most ofits dissolved components, such as certain by-products of theglucosyltransferase reaction. A filtrate or supernatant herein may befrom a glucosyltransferase reaction that synthesizes insolublealpha-1,3-glucan in certain embodiments.

A soluble fraction herein can be processed, if desired. Examples ofprocessing herein include dilution, concentration, hydrolytic treatment,pH modification, salt modification, and/or buffer modification.Processing can also include deactivating (e.g., heat-deactivation) theglucosyltransferase enzyme(s) used in the glucosyltransferase reactionfrom which the soluble fraction is obtained. Concentration of a solublefraction can be performed using any method or equipment known in the artsuitable for concentrating a solution. For example, a soluble fractioncan be concentrated by evaporation, such as with a rotary evaporator(e.g., temperature of about 40-50° C.). Other suitable types ofevaporation equipment include forced circulation or falling filmevaporators. A soluble fraction herein can be concentrated down to avolume that is about, or less than about, 75%, 80%, 85%, 90%, or 95%,for example, of the original soluble fraction volume. A concentratedsoluble fraction (e.g., concentrated filtrate) can optionally bereferred to as a syrup.

A soluble fraction herein can optionally be processed using a hydrolytictreatment. A hydrolytic treatment can be an enzymatic treatment in whichthe soluble fraction is treated with one or more hydrolytic enzymes, forexample. A hydrolytic enzyme can be one that hydrolyzes one or moreby-products (e.g., leucrose) of a glucosyltransferase reaction, forexample. Examples of useful hydrolytic enzymes herein includealpha-glucosidases such as transglucosidases (EC 2.4.1.24) (“EC” refersto Enzyme Commission number) and glucoamylases (EC 3.2.1.3). Methods oftreating a soluble fraction of a glucosyltransferase reaction with anyof these enzymes are disclosed in U.S. Patent Appl. Publ. Nos.2015/0240278 and 2015/0240279, which are incorporated herein byreference.

A soluble fraction herein can be unprocessed, if desired. An unprocessedsoluble fraction is one in which the fraction (or portion of a fraction)is isolated from a glucosyltransferase reaction and used in thedisclosed method without any sort of modification/processing afterisolating the soluble fraction. Examples of an unprocessed solublefraction include neat filtrate and neat supernatant.

A soluble fraction in certain preferred embodiments of the presentdisclosure is from an alpha-1,3-glucan synthesis reaction; such asoluble fraction is optionally a filtrate. A soluble fraction of analpha-1,3-glucan synthesis reaction herein comprises at least water,fructose and one or more types of saccharide (leucrose and/orgluco-oligosaccharides such as DP2-DP7). Other components that may be inthis type of soluble fraction include sucrose (i.e., residual sucrosenot consumed in the glucosyltransferase reaction), one or moreglucosyltransferase enzymes, glucose, buffer, salts, FermaSure®,borates, sodium hydroxide, hydrochloric acid, cell lysate components,proteins and/or nucleic acids, for example. Minimally, the components ofa soluble fraction from an alpha-1,3-glucan synthesis reaction hereininclude water, fructose, glucose, and one or more types ofoligosaccharides (leucrose and/or gluco-oligosaccharides such asDP2-DP7, optionally sucrose), for example.

It should be understood that the exact composition of sugars and othermaterial in a soluble fraction of a glucosyltransferase reaction is notbelieved to be critical for use as a source of oligosaccharides in amethod herein. It should also be understood that the ratio of sugars towater (i.e., wt % dry solids), which can be calculated by dividing themass of starting sugar to total initial reaction solution weight, can beadjusted either by evaporating water, preferably at temperatures below50° C. under vacuum, or addition of water, without significant impact tothe relative distribution of sugars in a soluble fraction of aglucosyltransferase reaction. It is also possible to increase thepercentage of sucrose in a soluble fraction by stopping theglucosyltransferase reaction before complete conversion (to glucan) isachieved, either by reducing the pH below the active range of theglucosyltransferase or by thermal deactivation of theglucosyltransferase.

Step (b) of a method herein embodies a glucosyltransferase reaction.Step (a) of providing oligosaccharides is performed before step (b).Thus, the oligosaccharides of step (a) are not provided by virtue oftheir possible in situ synthesis during step (b). In other words,performing a glucosyltransferase reaction alone in whicholigosaccharides are produced as a by-product does not in-and-of-itselfconstitute performing steps (a) and (b); oligosaccharides must bephysically (manually and/or mechanically) added to theglucosyltransferase reaction of step (b) in order to perform step (a).That being said, oligosaccharides produced by a glucosyltransferasereaction embodied by step (b) can be removed from that reaction(purified or unpurified, processed or unprocessed, as above; e.g., as afiltrate) and provided as oligosaccharides for step (a). In suchembodiments, steps (a) and (b) can be repeated one or more times, suchthat the oligosaccharides in each repeat of step (a) are provided fromthe products resulting from each immediately performed step (b). Steps(a) and (b) can be repeated 1, 2, 3, 4, 5, 6, or more times, forexample. Because of this repetition, methods following these embodimentscan optionally be referred to as continuous reaction processes and/oroligosaccharide recycling processes. In view of the foregoing, it shouldbe apparent that the glucosyltransferase reaction of step (a)(ii) insome methods herein can be the glucosyltransferase reaction embodied instep (b).

Alternatively, the glucosyltransferase reaction of step (a)(ii) in amethod herein can be different (distinct) from the glucosyltransferasereaction embodied in step (b). For example, oligosaccharides can beobtained from a first alpha-1,3-glucan synthesis reaction (e.g.,filtrate collected), after which the oligosaccharides are added to asecond alpha-1,3-glucan synthesis reaction that is distinct from thefirst reaction.

A glucosyltransferase enzyme is contacted with at least water, sucroseand added oligosaccharides in step (b) of a method herein. Examples ofsuitable glucosyltransferase enzymes are provided in the below Examples,and/or are disclosed in U.S. Pat. No. 7,000,000 and U.S. Pat. Appl.Publ. Nos. 2013/0244288, 2013/0244287, 2014/0087431, 2017/0002335 and2018/0072998 (all of which are incorporated herein by reference).

A glucosyltransferase enzyme in certain embodiments of the presentdisclosure comprises, or consists of, an amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to,or is 100% identical to, SEQ ID NO:2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 (or optionally any of these sequences without the startmethionine), for example, wherein the glucosyltransferase enzyme hasactivity. All these glucosyltransferases produce alpha-1,3-glucan with ahigh percentage of alpha-1,3 glycosidic linkages (≥95%) (refer to U.S.Appl. Publ. No. 2014/0087431, for example, which is incorporated hereinby reference).

SEQ ID NOs:16 (GTF 7527-short), 14 (GTF 2678), 9 (GTF 6855), 13 (GTF2919), and 11 (GTF 2765) each represent a glucosyltransferase that,compared to its respective wild type counterpart, lacks the signalpeptide domain and all or a substantial portion of the variable domain.Thus, each of these glucosyltransferase enzymes has a catalytic domainfollowed by a glucan-binding domain. The approximate location ofcatalytic domain sequences in these enzymes is as follows: 7527-short(residues 54-957 of SEQ ID NO:16), 2678 (residues 55-960 of SEQ IDNO:14), 6855 (residues 55-960 of SEQ ID NO:9), 2919 (residues 55-960 ofSEQ ID NO:13), 2765 (residues 55-960 of SEQ ID NO:11). The amino acidsequences of the approximate catalytic domains of GTFs 2678, 6855, 2919and 2765 have about 94.9%, 99.0%, 95.5% and 96.4% identity,respectively, with the approximate catalytic domain sequence of GTF7527-short (i.e., amino acids 54-957 of SEQ ID NO:16). All these fiveglucosyltransferase enzymes can produce alpha-1,3-glucan with about 100%alpha-1,3 linkages and a DPw of at least 400 (data not shown, refer toTable 4 of U.S. Pat. Appl. Publ. No. 2017/0002335, which is incorporatedherein by reference). Thus, a glucosyltransferase enzyme in certainembodiments can comprise, or consist of, a glucosyltransferase catalyticdomain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,98.5%, 99%, or 99.5% identical to, or is 100% identical to, the aminoacid sequence of a catalytic domain of GTF 7527-short, 2678, 6855, 2919,or 2765 (e.g., as listed above).

Although it is believed that a glucosyltransferase enzyme herein needonly have a catalytic domain sequence, such as one described above, theglucosyltransferase enzyme can be comprised within a larger amino acidsequence. For example, the catalytic domain may be linked at itsC-terminus to a glucan-binding domain, and/or linked at its N-terminusto a variable domain and/or signal peptide.

Still further examples of glucosyltransferase enzymes can be any asdisclosed herein and that include 1-300 (or any integer there between[e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on theN-terminus and/or C-terminus. Such additional residues may be from acorresponding wild type sequence from which the glucosyltransferaseenzyme is derived, or may be a heterologous sequence such as an epitopetag (at either N- or C-terminus) or a heterologous signal peptide (atN-terminus), for example. A glucosyltransferase enzyme herein typicallylacks an N-terminal signal peptide.

A glucosyltransferase enzyme in certain embodiments does not occur innature (i.e., non-native). For example, an enzyme herein is not believedto be one that is naturally secreted (i.e., mature form) from a microbe(from which the glucosyltransferase enzyme herein could possibly havebeen derived). A non-native enzyme in certain aspects comprises at leastone, two, or three amino acid(s) modified/substituted as compared to itsnative counterpart. The amino acid sequence of a glucosyltransferaseenzyme in certain aspects has been modified such that the enzymeproduces more products (alpha-1,3-glucan and fructose), and lessby-products (e.g., glucose, oligosaccharides such as leucrose), from agiven amount of sucrose substrate. For example, one, two, three, or moreamino acid residues of the catalytic domain of a glucosyltransferaseherein can be modified/substituted to obtain an enzyme that producesmore products (alpha-1,3-glucan and fructose). Suitable examples of sucha modified glucosyltransferase enzyme are disclosed in U.S. Pat. Appl.Publ. No. 2018/0072998, which is incorporated herein by reference.

A glucosyltransferase enzyme herein can be derived from any microbialsource, such as a bacteria. Examples of bacterial glucosyltransferaseenzymes are those derived from a Streptococcus species, Leuconostocspecies or Lactobacillus species. Examples of Streptococcus speciesinclude S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S.mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples ofLeuconostoc species include L. mesenteroides, L. amelibiosum, L.argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L.fructosum. Examples of Lactobacillus species include L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L.reuteri.

A glucosyltransferase enzyme can produce alpha-1,3-glucan as disclosedherein. For example, a glucosyltransferase enzyme can producealpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and aDPw of at least 100. The glucosyltransferase enzyme in certainembodiments does not have, or has very little (e.g., less than 1%),dextransucrase, reuteransucrase, or alternansucrase activity.

A glucosyltransferase enzyme herein can be prepared by fermentation ofan appropriately engineered microbial strain, for example. Recombinantenzyme production by fermentation is well known in the art usingmicrobial strains such as E. coli, Bacillus strains (e.g., B. subtilis),Ralstonia eutropha, Pseudomonas fluorescens, Saccharomyces cerevisiae,Pichia pastoris, Hansenula polymorpha, and species of Aspergillus (e.g.,A. awamori) and Trichoderma (e.g., T. reesei) (e.g., see Adrio andDemain, Biomolecules 4:117-139, which is incorporated herein byreference). A nucleotide sequence encoding a glucosyltransferase enzymeamino acid sequence is typically linked to a heterologous promotersequence to create an expression cassette for the enzyme. Such anexpression cassette may be incorporated on a suitable plasmid orintegrated into the microbial host chromosome, using methods well knownin the art. The expression cassette may include a transcriptionalterminator nucleotide sequence following the amino acid coding sequence.The expression cassette may also include, between the promoter sequenceand amino acid coding sequence, a nucleotide sequence encoding a signalpeptide that is designed to direct secretion of the glucosyltransferaseenzyme. At the end of fermentation, cells may be ruptured accordinglyand the glucosyltransferase enzyme can be isolated using methods such asprecipitation, filtration, and/or concentration. Alternatively, a lysatecomprising a glucosyltransferase can be used without further isolation.The activity of a glucosyltransferase enzyme can be confirmed bybiochemical assay, such as measuring its conversion of sucrose to glucanpolymer.

A glucosyltransferase enzyme herein can be primer-independent orprimer-dependent. Primer-independent glucosyltransferase enzymes do notrequire the presence of a primer to perform glucan synthesis. Aprimer-dependent glucosyltransferase enzyme requires the presence of aninitiating molecule in the reaction solution to act as a primer for theenzyme during glucan polymer synthesis. The term “primer” as used hereinrefers to any molecule that can act as the initiator for aglucosyltransferase enzyme. Primers that can be used in certainembodiments (in addition to added oligosaccharides as described herein,which are believed to serve as primers) include dextran. Dextran for useas a primer can be dextran T10 (i.e., dextran having a molecular weightof 10 kD), for example.

The activity of a glucosyltransferase enzyme herein can be determinedusing any method known in the art. For example, glucosyltransferaseenzyme activity can be determined by measuring the production ofreducing sugars (fructose and glucose) in a reaction solution containingsucrose (50 g/L), dextran T10 (1 mg/mL) and potassium phosphate buffer(pH 6.5, 50 mM), where the solution is held at 22-25° C. for 24-30hours. The reducing sugars can be measured by adding 0.01 mL of thereaction solution to a mixture containing 1 N NaOH and 0.1%triphenyltetrazolium chloride and then monitoring the increase inabsorbance at OD_(480nm) for five minutes.

Insoluble alpha-1,3-glucan is produced in the methods/reactions of thepresent disclosure. Alpha-1,3-glucan in certain aspects has at least 50%alpha-1,3 glycosidic linkages and a DPw of at least 100.

Alpha-1,3-glucan herein typically comprises at least 50%alpha-1,3-glycosidic linkages. In certain embodiments, at least about50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% (or any integer between 50% and 100%) of the constituentglycosidic linkages of an alpha-1,3-glucan are alpha-1,3 linkages. Insome embodiments, accordingly, alpha-1,3-glucan has less than about 50%,40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0% (orany integer value between 0% and 50%) glycosidic linkages that are notalpha-1,3. Typically, the linkages that are not alpha-1,3 are mostly orentirely alpha-1,6. It should be understood that the higher thepercentage of alpha-1,3 linkages present in alpha-1,3-glucan, thegreater the probability that the alpha-1,3-glucan is linear, since thereare lower occurrences of certain linkages forming branch points in thepolymer. Thus, alpha-1,3-glucan with 100% alpha-1,3 linkages is believedto be completely linear. In certain embodiments, alpha-1,3-glucan has nobranch points or less than about 5%, 4%, 3%, 2%, or 1% branch points asa percent of the glycosidic linkages in the polymer. Examples of branchpoints include alpha-1,6, -1,2 and -1,4 branch points.

Alpha-1,3-glucan herein can have a molecular weight in DPw or DPn of atleast about 100 in some aspects. DPw or DPn in some embodiments can beat least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1100, or 1200 (or any integerbetween 100 and 1200).

Alpha-1,3-glucan herein is insoluble in non-caustic aqueous systems,such as those conditions of a glucosyltransferase reaction herein (e.g.,pH 4-8, see below). In general, the solubility of a glucan polymer inaqueous settings herein is related to its linkage profile, molecularweight, and/or degree of branching. For example, alpha-1,3-glucan with≥95% 1,3 linkages is generally insoluble at a DP_(w) of 8 and above inaqueous conditions at 20° C. In general, as molecular weight increases,the percentage of alpha-1,3 linkages required for alpha-1,3-glucaninsolubility decreases.

In some other embodiments, an insoluble alpha-1,3-glucan can comprise atleast about 30% alpha-1,3 linkages and a percentage of alpha-1,6linkages that brings the total of both the alpha-1,3 and -1,6 linkagesin the alpha-1,3-glucan to 100%. For example, the percentage ofalpha-1,3 and -1,6 linkages can be about 30-40% and 60-70%,respectively. Glucosyltransferases for producing such alpha-1,3-glucanare disclosed in U.S. Pat. Appl. Publ. No. 2015/0232819, which isincorporated herein by reference. Alpha-1,3-glucan in these embodimentsdoes not comprise alternan (alternating 1,3 and 1,6 linkages).

The disclosed method comprises, in step (b), contacting at least water,sucrose, a glucosyltransferase enzyme, and oligosaccharides (as providedin step [a]). This contacting step can optionally be characterized asproviding a glucosyltransferase reaction composition comprising water,sucrose, a glucosyltransferase enzyme, and oligosaccharides. Thecontacting step of the disclosed method can be performed in any numberof ways. For example, a desired amount of sucrose can first be dissolvedor mixed in water, as well as the added oligosaccharides (optionally,other components can also be added at this stage of preparation, such asbuffer components), followed by addition of a glucosyltransferaseenzyme. The solution can be kept still, or agitated via stirring ororbital shaking, for example.

The temperature of a reaction composition herein can be controlled, ifdesired, and can be about 5-50° C., 20-40° C., 20-30° C., 20-25° C., forexample. In some aspects, the temperature can be about 5-15° C. (e.g.,˜8-12° C., ˜9-11° C., ˜10° C.), 15-25° C. (e.g., ˜20° C.), or 25-35° C.(e.g., ˜30° C.).

Oligosaccharides herein can be provided such that their initialconcentration in a glucosyltransferase reaction set up in step (b) is atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 5-10, or 5-15g/L, for example. The foregoing concentrations can be provided usingpurified or unpurified (e.g., filtrate) oligosaccharide compositions. An“initial concentration” of oligosaccharides herein can, for example,refer to the oligosaccharide concentration in a glucosyltransferasereaction just after a minimum set of reaction components have beenadded/combined (at least water, sucrose, glucosyltransferase enzyme,optionally oligosaccharides). Oligosaccharides can be added to aglucosyltransferase reaction in batch or fed-batch mode. In batch mode,oligosaccharides are all added (are all present) at the beginning of, orwithin about 10-15 minutes of starting, a reaction, whereasoligosaccharides are added throughout a reaction in fed-batch mode. Forexample, a fed-batch can comprise adding oligosaccharides continuouslyor incrementally (e.g., dosing every 30 or 60 minutes) throughout,and/or during a period of (e.g., first 6 hours), a reaction. The totalamount of oligosaccharides provided in a fed-batch mode reaction can bethe same as the amount provided via any of the initial concentrationslisted above. Oligosaccharides in some embodiments are added to aglucosyltransferase reaction either at its beginning or within 1-2 hoursof its beginning.

The initial concentration of sucrose in a reaction composition hereincan be about 20-400 g/L, 75-175 g/L, or 50-150 g/L, for example. In someaspects, the initial sucrose concentration is at least about 50, 75,100, 150 or 200 g/L, or is about 50-600 g/L, 100-500 g/L, 50-100 g/L,100-200 g/L, 150-450 g/L, 200-450 g/L, or 250-600 g/L. “Initialconcentration of sucrose” refers to the sucrose concentration in aglucosyltransferase reaction composition just after all the reactionsolution components have been added/combined (at least water, sucrose,glucosyltransferase enzyme, optionally added oligosaccharides).

Sucrose used in a glucosyltransferase reaction solution can be highlypure (≥99.5%) or be of any other purity or grade. For example, sucrosecan have a purity of at least 99.0%, or can be reagent grade sucrose. Asanother example, incompletely refined sucrose can be used. Incompletelyrefined sucrose herein refers to sucrose that has not been processed towhite refined sucrose. Thus, incompletely refined sucrose can becompletely unrefined or partially refined. Examples of unrefined sucroseare “raw sucrose” (“raw sugar”) and solutions thereof. Examples ofpartially refined sucrose have not gone through one, two, three, or morecrystallization steps. Sucrose herein may be derived from any renewablesugar source such as sugar cane, sugar beets, cassava, sweet sorghum, orcorn. Suitable forms of sucrose useful herein are crystalline form ornon-crystalline form (e.g., syrup, cane juice, beet juice), for example.Additional suitable forms of incompletely refined sucrose are disclosedin U.S. Pat. Appl. Publ. No. 2015/0275256, which is incorporated hereinby reference. The ICUMSA (International Commission for Uniform Methodsof Sugar Analysis) of incompletely refined sucrose herein can be greaterthan 150, for example. Methods of determining ICUMSA values for sucroseare disclosed, for example, by the International Commission for UniformMethods of Sugar Analysis in ICUMSA Methods of Sugar Analysis: Officialand Tentative Methods Recommended by the International Commission forUniform Methods of Sugar Analysis (ICUMSA) (Ed. H. C. S. de Whalley,Elsevier Pub. Co., 1964), which is incorporated herein by reference.ICUMSA can be measured in some aspects by ICUMSA Method GS1/3-7 asdescribed by R. J. McCowage, R. M. Urquhart and M. L. Burge(Determination of the Solution Colour of Raw Sugars, Brown Sugars andColoured Syrups at pH 7.0—Official, Verlag Dr Albert Bartens, 2011revision), which is incorporated herein by reference.

The pH of a reaction composition in certain embodiments can be about4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-8.0, 5.5-7.5, or 5.5-6.5. In someaspects, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or8.0. The pH can be adjusted or controlled by the addition orincorporation of a suitable buffer, including but not limited to:phosphate, tris, acetate, citrate, or any combination thereof. Thebuffer concentration in a reaction composition herein can be about0.1-300 mM, 0.1-100 mM, 10-100 mM, 10 mM, 20 mM, or 50 mM, for example.A suitable amount of DTT (dithiothreitol, e.g., about 1.0 mM) canoptionally be added to a reaction solution.

A glucosyltransferase reaction can be contained within any vessel (e.g.,an inert vessel/container) suitable for applying one or more of thereaction conditions disclosed herein. An inert vessel in some aspectscan be of stainless steel, plastic, or glass (or comprise two or more ofthese components) and be of a size suitable to contain a particularreaction. For example, the volume/capacity of an inert vessel (and/orthe volume of a reaction composition herein), can be about, or at leastabout, 1, 10, 50, 100, 500, 1000, 2500, 5000, 10000, 12500, 15000, or20000 liters An inert vessel can optionally be equipped with a stirringdevice.

A reaction composition herein can contain one, two, or moreglucosyltransferase enzymes, for example. In some embodiments, only oneor two glucosyltransferase enzymes is/are comprised in a reactioncomposition. A glucosyltransferase reaction herein can be, and typicallyis, cell-free (e.g., no whole cells present).

Completion of a reaction in certain embodiments can be determinedvisually (e.g., no more accumulation of insoluble glucan), and/or bymeasuring the amount of sucrose left in the solution (residual sucrose),where a percent sucrose consumption of at least about 90%, 95%, or 99%can indicate reaction completion. In some aspects, a reaction can beconsidered complete when its sucrose content is at or below about 5 g/L.A reaction of the disclosed process can be conducted for about 1 hour toabout 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 96,120, 144, or 168 hours, for example. A reaction can optionally beterminated and/or otherwise treated to stop glucosyltransferase activityby heating it to at least about 65° C. for at least about 30-60 minutes.

The yield of alpha-1,3-glucan produced in a glucosyltransferase reactionherein can be about, at least about, or up to about, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%,65%, 70%, 75%, 80% or 85%, for example, based on the weight or moles ofsucrose converted in the reaction, or based on the glucosyl component ofthe reaction. Such a yield in some aspects is achieved in a reactionconducted for about 16-24 hours (e.g., ˜20 hours), and/or is as measuredusing HPLC or NIR spectroscopy.

Alpha-1,3-glucan produced in a method in certain embodiments mayoptionally be isolated. In certain embodiments, isolating insolublealpha-1,3-glucan can include at least conducting a step ofcentrifugation and/or filtration. Isolation can optionally furthercomprise washing alpha-1,3-glucan one, two, or more times with water orother aqueous liquid, and/or drying the alpha-glucan product.

An isolated alpha-1,3-glucan product herein, as provided in a dry form,can comprise no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01wt % water, for example. In some aspects, an alpha-1,3-glucan product isprovided in an amount of at least 1 gram (e.g., at least about 2.5, 5,10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000,50000, or 100000 g); such an amount can be a dry amount, for example.

Examples of conditions and/or components suitable for synthesizinginsoluble alpha-1,3-glucan herein are disclosed in U.S. Pat. No.7,000,000, and U.S. Pat. Appl. Publ. Nos. 2013/0244288, 2013/0244287,2013/0196384, 2013/0157316, 2015/0275256, 2015/0240278, 2015/0240279,2014/0087431, 2017/0002335 and 2018/0072998, all of which areincorporated herein by reference.

Any of the disclosed conditions for synthesizing insolublealpha-1,3-glucan, such as the foregoing or those described in the belowExamples, can be applied to practicing a reaction composition aspresently disclosed (and vice versa).

The present disclosure also concerns a reaction composition forproducing insoluble alpha-1,3-glucan. This reaction compositioncomprises at least water, sucrose, a glucosyltransferase enzyme thatsynthesizes insoluble alpha-1,3-glucan, and oligosaccharides. Theoligosaccharides are added during preparation of the reactioncomposition and:

-   -   (i) comprise alpha-1,3 and alpha-1,6 glycosidic linkages, and/or    -   (ii) are derived from a glucosyltransferase reaction.        Insoluble alpha-1,3-glucan is produced in the reaction        composition.

A reaction composition herein can be practiced following any of thepresently disclosed embodiments or below Examples regarding methods ofproducing insoluble alpha-1,3-glucan, for example. Thus, any of featuresof such embodiments can characterize embodiments of a reactioncomposition herein.

In certain embodiments, the yield of alpha-1,3-glucan produced by aglucosyltransferase reaction can be increased compared to the yield ofalpha-1,3-glucan that would be produced if step (b) is performed withoutadded oligosaccharides (i.e., without the oligosaccharides of step [a]).For example, the yield of alpha-1,3-glucan produced can be increased byat least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 110%, or 120% compared to the yield ofalpha-1,3-glucan that would be produced if step (b) lacked addedoligosaccharides. It would be understood that percent increase ofalpha-1,3-glucan product yield in a method herein can be measured, ifdesired, with respect to a suitable control glucosyltransferase reaction(e.g., a reaction having the same parameters as in step [b], except forthe addition of oligosaccharides). In some aspects, an increase in yieldcharacterizes a reaction comprising a glucosyltransferase that does nothave any catalytic domain amino acid substitutions as compared to itscorresponding native amino acid sequence.

The relative reaction rate of the glucosyltransferase reaction of step(b) in certain embodiments can be increased compared to the reactionrate that would be observed if step (b) was performed without theoligosaccharides provided in step (a). For example, the relativereaction rate of the glucosyltransferase reaction of step (b) can be atleast about 1.025, 1.05, 1.075, 1.10, 1.15, 1.20, 1.25, 1.30, 1.40,1.50, 1.60, 1.70, 1.80, 1.90, 2.00, or 2.10 with respect to the reactionrate of a suitable control glucosyltransferase reaction. To illustrate,if the relative reaction rate of a reaction herein is at least about1.25 with respect to a control reaction, the reaction rate of thisreaction is at least about 25% higher than the reaction rate of thecontrol reaction. The reaction rate of a reaction can be expressed interms of the change in concentration/amount of reactant(s) (e.g.,sucrose) and/or the change in concentration/amount of product(s) (e.g.,alpha-1,3-glucan) per unit time per unit concentration of activeglucosyltransferase enzyme. Reaction rates can be measured, for example,in grams alpha-1,3-glucan produced per liter per hour (g L⁻¹ h⁻¹).

By-product formation can optionally be reduced in theglucosyltransferase reaction of step (b) of a method herein of producinginsoluble alpha-1,3-glucan, compared to the by-product formation thatwould be observed if step (b) was performed without the oligosaccharidesprovided in step (a). For example, the amount of glucose, leucrose,and/or gluco-oligosaccharide by-products formed in step (b) can bereduced compared to a suitable control glucosyltransferase reaction.Such reduction can be by at least about 5%, 10%, 15%, 20%, 25%, 30%,40%, 50%, or 60%, for example.

In certain embodiments, the viscosity of alpha-1,3-glucan produced by aglucosyltransferase reaction can be decreased compared to the viscosityof alpha-1,3-glucan that would be produced if step (b) is performedwithout added oligosaccharides (i.e., without the oligosaccharides ofstep [a]). This viscosity can be determined for alpha-1,3-glucan asmixed or dissolved in a liquid. The viscosity of an alpha-1,3-glucanproduct herein can be at least about 5%, 10%, 15%, 20%, or 25% less, forexample, than the viscosity of alpha-1,3-glucan that would be producedif step (b) lacked added oligosaccharides. It would be understood thatpercent decrease of alpha-1,3-glucan product viscosity in a methodherein can be measured, if desired, with respect to a suitable controlglucosyltransferase reaction (e.g., a reaction having the sameparameters as in step [b], except for the addition of oligosaccharides).

The viscosity of alpha-1,3-glucan herein can be determined as mixed ordissolved in a liquid. In certain aspects, such a determination can bemade with alpha-1,3-glucan as mixed in an aqueous liquid such as wateror aqueous solution that is not caustic (e.g., a non-caustic aqueousliquid can have a pH of about 4-10, 5-9, 6-8, or 7); mixing isrecommended since alpha-1,3-glucan herein is typically insoluble in suchaqueous conditions. This mixing can be performed using any suitablemeans for effectively mixing alpha-1,3-glucan in a non-caustic aqueousliquid, such as homogenization or microfluidization (e.g., as disclosedin any of International Pat. Appl. Publ. No. WO2016/126685 or U.S. Pat.Appl. Publ. Nos. 2015/0167243, 2005/0249853 2003/0153746 and2018/0021238, which are all incorporated herein by reference), forexample. Mixing of alpha-1,3-glucan in a non-caustic aqueous liquid cantypically be done to prepare an aqueous slurry and/or dispersion(colloidal dispersion) of the glucan. The viscosity of such aqueouscompositions can optionally be measured as slurry viscosity, in units ofcP, at a shear rate of about 5-250 s⁻¹ (e.g., 7-200 s⁻¹), at atemperature of about 15-25° C. (e.g., ˜20° C.), and/or with a 2-10 wt %(e.g., ˜4-5 wt %) alpha-1,3-glucan aqueous mixture.

In certain aspects, a viscosity determination can be made withalpha-1,3-glucan as dissolved in a liquid. Such a liquid can be acaustic aqueous solution having a pH of at least about 11, for instance.A caustic aqueous solution can comprise at least a hydroxide (e.g.,NaOH, KOH, tetraethyl ammonium hydroxide), and/or be as disclosed inInternational Pat. Appl. Publ. Nos. WO2015/200612 or WO2015/200590, orU.S. Pat. Appl. Publ. Nos. 2017/0208823 or 2017/0204203 (all of whichare incorporated herein by reference), for example. In some aspects, aliquid for dissolving alpha-1,3-glucan herein for measuring viscositycan be non-aqueous such as one comprising an organic solvent (e.g.,organic ionic liquid). Examples of a suitable organic solvent herein cancomprise N,N-dimethylacetamide (DMAc) (optionally with about 0.5%-5%LiCl), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), pyridine,SO₂/diethylamine (DEA)/DMSO, LiCl/1,3-dimethyl-2-imidazolidinone (DMI),DMSO/tetrabutyl-ammonium fluoride trihydrate (TBAF),N-methylpyrrolidone, and/or N-methylmorpholine-N-oxide (NMMO). In someaspects, alpha-1,3-glucan can be dissolved to a concentration of about5-15 mg/mL (e.g., 10 mg/mL) in a suitable organic solvent such asDMAc/0.5% LiCl for measuring viscosity. The viscosity of dissolvedalpha-1,3-glucan herein can be measured as intrinsic viscosity (IV,symbolized as “η”, provided in units of mL/g) in some embodiments. IVmeasurements herein can be obtained, for example, using any suitablemethod such as disclosed in U.S. Pat. Appl. Publ. Nos. 2017/0002335 and2017/0002336, Weaver et al. (J. Appl. Polym. Sci. 35:1631-1637), or Chunand Park (Macromol. Chem. Phys. 195:701-711), which are all incorporatedherein by reference.

The present disclosure also concerns a composition comprising insolublealpha-1,3-glucan produced according to any method herein of producinginsoluble alpha-1,3-glucan. In certain embodiments, such a compositioncan be either an aqueous composition or a non-aqueous composition. Insome aspects, the viscosity of an insoluble alpha-1,3-glucan productherein is less than the viscosity of insoluble alpha-1,3-glucan (controlglucan) that would have been produced if the oligosaccharides were notprovided in the method/reaction. Viscosity can be measured with anymethodology as described above with respect to alpha-1,3-glucan as mixedor dissolved in a liquid. The viscosity of the present alpha-1,3-glucanproduct can be at least about 5%, 10%, 15%, 20%, or 25% less than theviscosity of the control glucan, for example. The viscosity/DPwrelationship of the present alpha-1,3-glucan product can be, forexample, as disclosed in the below Examples showing that addinggluco-oligosaccharides to a glucosyltransferase reaction reducesviscosity.

Non-limiting examples of compositions and methods disclosed hereininclude:

-   1. A method for producing insoluble alpha-1,3-glucan comprising:-   (a) providing oligosaccharides that:    -   (i) comprise alpha-1,3 and alpha-1,6 glycosidic linkages, and/or    -   (ii) are produced from a glucosyltransferase reaction;-   (b) contacting at least water, sucrose, a glucosyltransferase enzyme    that synthesizes insoluble alpha-1,3-glucan, and the    oligosaccharides, wherein insoluble alpha-1,3-glucan is produced;    and-   (c) optionally, isolating the insoluble alpha-1,3-glucan produced in    step (b).-   2. The method of embodiment 1, wherein the oligosaccharides comprise    about 60-99% alpha-1,3 and about 1-40% alpha-1,6 glycosidic    linkages.-   3. The method of embodiment 1 or 2, wherein the oligosaccharides    have a degree of polymerization (DP) of 2 to 10.-   4. The method of embodiment 1, 2, or 3, wherein the oligosaccharides    are purified or unpurified.-   5. The method of embodiment 4, wherein the oligosaccharides are    produced from the glucosyltransferase reaction of (a)(ii).-   6. The method of embodiment 5, wherein the glucosyltransferase    reaction of (a)(ii) synthesizes insoluble alpha-1,3-glucan.-   7. The method of embodiment 5 or 6, wherein the oligosaccharides are    provided as a soluble fraction of the glucosyltransferase reaction    of (a)(ii), and wherein the soluble fraction is processed or    unprocessed.-   8. The method of embodiment 7, wherein the soluble fraction is a    portion of, or all of, a filtrate of the glucosyltransferase    reaction of (a)(ii).-   9. The method of embodiment 1, 2, 3, 4, 5, 6, 7, or 8, wherein the    oligosaccharides are provided in step (b) at an initial    concentration of at least about 1 g/L.-   10. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein    the yield of insoluble alpha-1,3-glucan produced is increased    compared to the yield of insoluble alpha-1,3-glucan that would be    produced if step (b) lacked the oligosaccharides.-   11. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,    wherein the viscosity of the insoluble alpha-1,3-glucan produced is    decreased compared to the viscosity of insoluble alpha-1,3-glucan    that would be produced if step (b) lacked the oligosaccharides,    wherein viscosity is measured with alpha-1,3-glucan as mixed or    dissolved in a liquid.-   12. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11,    wherein the insoluble alpha-1,3-glucan produced has at least 50%    alpha-1,3 glycosidic linkages and a weight-average degree of    polymerization (DPw) of at least 100.-   13. The method of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or    13, wherein steps (a) and (b) are repeated one or more times, and    wherein the oligosaccharides in each repeated step (a) are provided    from the products (by-products) resulting from each immediately    preceding step (b).-   14. A reaction composition for producing insoluble alpha-1,3-glucan,    the reaction composition comprising at least water, sucrose, a    glucosyltransferase enzyme that synthesizes insoluble    alpha-1,3-glucan, and oligosaccharides, wherein the oligosaccharides    are added during preparation of the reaction composition and:    -   (i) comprise alpha-1,3 and alpha-1,6 glycosidic linkages, and/or    -   (ii) are produced from a glucosyltransferase reaction,        wherein insoluble alpha-1,3-glucan is produced in the reaction        composition, optionally wherein the reaction composition is        characterized by any feature of any one of embodiments 2-13.-   15. A composition comprising insoluble alpha-1,3-glucan produced    according to the method of any one of embodiments 1-13, or produced    in the reaction composition of embodiment 14.-   16. The composition of embodiment 15, wherein the viscosity of the    insoluble alpha-1,3-glucan is less than the viscosity of insoluble    alpha-1,3-glucan that would have been produced if the    oligosaccharides are not provided in the method or reaction    composition, wherein viscosity is measured with alpha-1,3-glucan as    mixed or dissolved in a liquid.

EXAMPLES

The present disclosure is further exemplified in the following Examples.It should be understood that these Examples, while indicating certainpreferred aspects herein, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the disclosed embodiments,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt the disclosed embodiments tovarious uses and conditions.

General Methods

All reagents were obtained from Sigma-Aldrich (St. Louis, Mo.) unlessstated otherwise. Sucrose was obtained from VWR (Radnor, Pa.).

Preparation of Crude Extracts of Glucosyltransferase (GTF) Enzymes

The Streptococcus salivarius GTFJ enzyme (SEQ ID NO:2) used in certainof the following examples was expressed in E. coli strain DH10B using anisopropyl beta-D-1-thiogalactopyranoside (IPTG)-induced expressionsystem. SEQ ID NO:2 has an N-terminal 42-residue deletion compared tothe S. salivarius GTFJ amino acid sequence in GENBANK Identification No.47527. Briefly, E. coli DH10B cells were transformed to express SEQ IDNO:2 from a DNA sequence (SEQ ID NO:1) codon-optimized for expression inE. coli. This DNA sequence was contained in the expression vector,pJexpress404® (DNA 2.0, Menlo Park Calif.). The transformed cells wereinoculated to an initial optical density (OD at 600 nm) of 0.025 in LBmedium (10 g/L Tryptone; 5 g/L yeast extract, 10 g/L NaCl) and allowedto grow at 37° C. in an incubator while shaking at 250 rpm. The cultureswere induced by addition of 1 mM IPTG when they reached an OD₆₀₀ of0.8-1.0. Induced cultures were left on the shaker and harvested 3 hourspost induction.

GTFJ enzyme (SEQ ID NO:2) was harvested by centrifuging cultured cells(25° C., 16,000 rpm) in an Eppendorf® centrifuge, re-suspending thecells in 5.0 mM phosphate buffer (pH 7.0) and cooling to 4° C. on ice.The cells were broken using a bead beater with 0.1-mm silica beads, andthen centrifuged at 16,000 rpm at 4° C. to pellet the unbroken cells andcell debris. The crude extract (containing soluble GTFJ enzyme, SEQ IDNO:2) was separated from the pellet and analyzed by Bradford proteinassay to determine protein concentration (mg/m L).

The GTF enzymes used in Example 5 were prepared as follows. E. coliTOP10® cells (Invitrogen, Carlsbad Calif.) were transformed with apJexpress404®-based construct containing a particular GTF-encoding DNAsequence. Each sequence was codon-optimized to express the GTF enzyme inE. coli. Individual E. coli strains expressing a particular GTF enzymewere grown in LB medium with ampicillin (100 mg/mL) at 37° C. withshaking to OD₆₀₀=0.4-0.5, at which time IPTG was added to a finalconcentration of 0.5 mM. The cultures were incubated for 2-4 hours at37° C. following IPTG induction. Cells were harvested by centrifugationat 5,000×g for 15 minutes and resuspended (20% w/v) in 50 mM phosphatebuffer pH 7.0 supplemented with DTT (1.0 mM). Resuspended cells werepassed through a French Pressure Cell (SLM Instruments, Rochester, N.Y.)twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30minutes at 12,000×g at 4° C. The resulting supernatant was analyzed bythe BCA protein assay and SDS-PAGE to confirm expression of the GTFenzyme, and the supernatant was stored at −20° C.

Analysis of Reaction Profiles

Periodic samples from reactions were taken and analyzed using anAgilent® 1260 HPLC equipped with a refractive index detector. An Aminex®HPX-87C column (BioRad, Hercules, Calif.) having deionized water at aflow rate of 0.6 mL/min and 85° C. was used to quantitate the level ofsucrose, glucose, leucrose and fructose in the reaction mixtures. AnAminex® HPX-42A column (BioRad) having deionized water at a flow rate of0.6 mL/min and 85° C. was used to quantitate soluble oligosaccharideby-products.

Analysis of Glucan Molecular Weight

Insoluble glucan polymer isolated from glucosyltransferase reactions wastreated with N,N-dimethylacetamide (DMAc) with 5% lithium chloride(LiCl) at 100° C. for 16 hours to form a glucan polymer solution. Thissolution (100 μL) was then injected into an Alliance™ 2695 HPLC (WatersCorporation, Milford, Mass.) equipped with a differential refractometerdetector operating at 50° C. The mobile phase (DMAc containing 0.11 wt %LiCl) passed at a flow rate of 0.5 mL/min through four styrene-divinylbenzene columns in series; specifically, one KD-802, one KD-801, and twolinear KD-806M columns (Shodex, Japan). The molecular weightdistribution of the glucan polymer sample was determined by comparisonof retention time to a broad glucan standard.

Example 1 Glucan Polymerization Reactions Using GTFJ Enzyme (SEQ IDNO:2)

This example discloses information on the conversion of sucrose toinsoluble alpha-1,3-glucan polymer and soluble sugars, and details howthe raw material utilized in Example 2 was generated.

Sucrose (3000 g) was added to a clean 5-gallon polyethylene bucket.Water (18.1 L) and Fermasure™ (10 mL) were added to the bucket, and thepH was adjusted to 7.0 by addition of 5 vol % NaOH and 5 vol % H₂SO₄.The final volume was ˜20 L and the initial concentration of sucrose asmeasured by HPLC was 152.5 g/L. The glucan polymerization reaction wasinitiated by adding 0.3 vol % of crude GTFJ enzyme (SEQ ID NO:2) extractprepared as described in the General Methods section. This extractcontained about 2.9 mg/mL of protein. Agitation to the reaction solutionwas provided using an overhead mechanical motor equipped with a glassshaft and PTFE blade.

After 48 hours, HPLC analysis revealed that 96% of the sucrose had beenconsumed and the reaction was deemed to be complete. Insolublealpha-1,3-glucan was removed by filtration, and the mother liquor(filtrate) was then concentrated using a rotary evaporator (bathtemperature of 40-50° C.) to a total sugar concentration of 320 g/Lsugars. The composition of the concentrated sugar solution is providedin Table 2.

TABLE 2 Composition of a Concentrated Filtrate of a Glucan SynthesisReaction Sucrose Leucrose Glucose Fructose DP2 DP3 DP4 DP5 DP6 DP7 Totalg/L 13.5 130.6 25.5 103.8 18.3 14.1 8.2 3.6 1.5 0.9 320.1 wt %^(a) 4.240.8 8 32.4 5.7 4.4 2.6 1.1 0.5 0.3 100 ^(a)Weight percentage is withrespect to the measured saccharide components.

Table 2 indicates that the concentrated filtrate obtained uponcompletion of the above glucan synthesis reaction contained saccharidesin which about 14-15 wt % thereof were oligosaccharide (DP2-DP7)by-products. This concentrated filtrate was used in Example 2 forchromatographic isolation of oligosaccharides.

Example 2 Isolation and Analysis of Oligosaccharides Using Ion-ExchangeResins

This example discloses how oligosaccharides were isolated from aconcentrated filtrate of a glucan synthesis reaction by chromatographicseparation, and analyzed for glycosidic linkage profile. These isolatedoligosaccharides were used in Examples 3, 5 and 7.

Chromatographic separation employing a strong acid cation-exchange resinwas used to isolate the oligosaccharide fraction of the concentratedfiltrate prepared in Example 1. The physical parameters of the columnused for this separation appear in Table 3.

TABLE 3 Physical Parameters of the Column Used for ChromatographicSeparation Resin Type FINEX CS11GC, #227 Ion form Na⁺ Crosslinking, %divinyl benzene  5% Particle size (mm) 0.34 Bed length (m) 1.64 Columndiameter (m) 0.093

The concentrated sugar solution (i.e., concentrated filtrate) preparedin Example 1 was filtered and diluted to 25 g dry solids/100 g solutionusing tap water. Prior to addition of the sugar solution to the columnresin, the resin was washed with six bed volumes (BV) of sodium chloridesolution (three BV at 10 wt % sodium chloride followed by three BV at 5wt % sodium chloride) to convert the resin to the sodium form. The sugarsolution (0.6 L) was then fed to the column, after which the column waseluted using water at a flow rate of 50 mL/min. The run conditions ofthe chromatographic separation are summarized in Table 4.

TABLE 4 Chromatographic Separation Run Conditions Feed size (L) 0.6 Feeddry solids (g/100 g) 25 Column temp (° C.) 65 Flow rate (mL/min) 50

An oligosaccharide solution eluted between 11 and 21 minutes. A smallamount of salts—indicated by an increase in conductivity—was eluted atthe same time. The oligosaccharide fraction thus prepared was analyzedby HPLC to determine its product distribution. In total, the fractioncontained >89% of oligosaccharides containing three or more hexose unitsand less than 1.5% of identifiable mono- and di-saccharides. Thisfraction was concentrated to a total dry weight of 317 g/L using a thinfilm evaporator (LCI Corporation, Charlotte, N.C.) followed by rotaryevaporation with a ROTAVAPOR (R-151; Buchi, New Castle, Del.). Theproduct distribution of the concentrated fraction as measured by HPLCappears in Table 5.

TABLE 5 Product Distribution of Concentrated Oligosaccharide FractionSucrose Leucrose Glucose Fructose DP2 DP3 DP4 DP5 DP6 DP7 Total g/L 0.02.5 0.0 0.7 31.5 75.9 101.8 62.1 26.9 15.3 316.7 wt %^(a) 0.0 0.8 0.00.2 9.9 23.9 32.1 19.6 8.5 4.8 100 ^(a)Weight percentage is with respectto the measured saccharide components.

The concentrated oligosaccharide solution of Table 5 was analyzed using¹H NMR. NMR data were acquired on an Agilent® DD2 spectrometer operatingat 500 MHz for ¹H using a 5-mm cryogenic triple-resonance pulsed-fieldgradient (PFG) probe. Water suppression was obtained by carefullyplacing the observe transmitter frequency on resonance for the residualwater signal in a “presat” experiment, and then using the first slice ofa NOESY experiment with a full phase cycle (multiple of 32) and a mixtime of 10 ms. One-dimensional ¹H spectra were acquired with a spectralwidth of 6410 Hz, acquisition time of 5.1 s, 65536 data points, 4 spresaturation and a 90-degree pulse of 5.85 μs. Sample temperature wasmaintained at 25° C. Chemical shift assignments for different anomericlinkages were taken from Goffin et al. (2009, Bull Korean Chem. Soc.30:2535-2541. Analysis of the spectra for this sample, which appears inFIG. 1 , reveals that the oligosaccharides comprised about 78% alpha-1,3and about 22% alpha-1,6 glycosidic linkages.

Thus, oligosaccharides from a concentrated filtrate of a glucansynthesis reaction were isolated and analyzed. The above concentratedoligosaccharide solution was used in Examples 3, 5 and 7.

Example 3 Comparison of Glucosyltransferase Reactions Lacking orContaining Added Oligosaccharides

This example discloses that adding purified oligosaccharides (Example 2)containing a significant fraction of DP2+ material to alpha-1,3-glucansynthesis reactions results in increased insoluble alpha-1,3-glucanproduct yields compared to reactions lacking such addedoligosaccharides. This example also demonstrates that this benefit(increased glucan product yield) is conferred across a wide variety ofreaction conditions and oligosaccharide loadings.

Glucan synthesis reactions were prepared as follows. Sucrose (10 g),0.27 g dihydrogen potassium phosphate (KH₂PO₄), 94 mL water, and 50micro-L Fermasure™ were added to a 125-mL clean glass bottles equippedwith a polypropylene cap. No oligosaccharides were added to thepreparation in comparative Example 3A (Table 6). In Examples 3.1 and 3.2(Table 6), certain amounts of the oligosaccharide solution prepared inExample 2 (Table 5) were added to each respective preparation; theamount of water added to each respective preparation was reduced by anequivalent volume. Each of the preparations contained a trace amount ofglucose that came primarily from the sucrose component; no additionalglucose was added to any of the preparations. Each preparation wasagitated in an incubator shaker (temperature-controlled to 25° C.) untila solution formed, at which point the pH of each preparation wasadjusted to 5.5 using 5 wt % aqueous sodium hydroxide or 5 wt % aqueoussulfuric acid. A sample of each preparation was taken for analysis byHPLC, after which 0.3 vol % of crude GTFJ enzyme (SEQ ID NO:2) extractprepared as described in the General Methods section was added to eachpreparation to initiate a polymerization reaction. Samples of eachreaction were periodically taken and analyzed by HPLC as the reactionsprogressed. The initial rate of reaction was calculated by the amount ofsucrose that was consumed in the first two hours of the polymerization.Once each reaction was deemed complete, insoluble polymer product wasisolated from the reaction by filtration, washed with 200 mL water,washed with 100 mL acetone, and then dried.

Results for each reaction are shown in Table 6, which demonstrate thatthe yield of insoluble alpha-1,3-glucan polymer product is increasedwhen oligosaccharides were added to the reaction (compare Examples 3.1and 3.2 with Example 3A). The results also demonstrate that the yield ofinsoluble alpha-1,3-glucan obtained was further improved upon adding anadditional amount of oligosaccharides (compare Example 3.2 with 3.1).

TABLE 6 Profiles of Glucosyltransferase Reactions Lacking or ContainingAdded Oligosaccharides Example 3A 3.1 3.2 Nominal sucrose (g/L) 100 100100 Actual sucrose (g/L) 97.2 104.6 107.1 Initial oligosaccharides, DP2+(g/L) 0.0 5.3 22.0 Initial glucose (g/L) 1.3 1.1 1.6 Initial rate (gsucrose consumed/L-hr) 5.3 14.1 12.3 % sucrose reacted 94.3 96.4 98.1Yield polymer (g/L) 15.4 24.6 27.5 Yield glucose (g/L) 8.1 5.9 4.9 Yieldoligomers (g/L) 8.2 8.9 24.6 Yield polymer (g/g sucrose reacted) 0.170.24 0.26 Yield glucose (g/g sucrose reacted) 0.075 0.048 0.031 Yieldoligomer (g/g sucrose reacted) 0.090 0.035 0.025

The benefits conferred upon adding oligosaccharides to analpha-1,3-glucan synthesis reaction were obtained over a range oftemperature and sucrose loadings. Reactions in Examples 3.3-3.5 wereprepared and carried out in the same manner as described above, exceptthat the initial sucrose concentration or temperature were modified. Theresults of these reactions, as well as those of the reactions ofcomparative Examples 3B, 3C and 3D (controls for Examples 3.3, 3.4 and3.5, respectively) which did not have any added oligosaccharides, aresummarized in Table 7.

TABLE 7 Profiles of Glucosyltransferase Reactions Lacking or ContainingAdded Oligosaccharides Performed under Different Sucrose or TemperatureConditions Example 3B 3.3 3C 3.4 3D 3.5 Nominal sucrose (g/L) 50 50 150150 100 100 Actual sucrose (g/L) 50.1 50.2 150.8 148.7 96.0 103.2Temperature (° C.) 25 25 25 25 37 37 Initial oligosaccharides, 0.0 6.00.0 5.8 0.0 5.8 DP2+ (g/L) Initial glucose (g/L) 0.0 0.4 0.0 1.2 0.0 0.8% sucrose reacted 96.9 99.5 95.8 97.6 97.0 97.2 Yield polymer (g/L) 9.514.2 23.0 33.4 21.7 27.5 Yield polymer (g/g sucrose 0.20 0.28 0.16 0.230.23 0.27 reacted)

Thus, adding oligosaccharide by-products of an alpha-1,3-glucansynthesis reaction to a new alpha-1,3-glucan synthesis reaction canincrease the yield of alpha-1,3-glucan produced by the new reaction andincrease the rate of the polymerization while simultaneously decreasingthe yield of unwanted oligomers and glucose. This reaction modulationoccurs over a variety of conditions.

Example 4 Insoluble Alpha-1,3-Glucan Yields ARE Reduced inGlucosyltransferase Reactions in Which Glucose is Added Instead ofOligosaccharides

This example discloses that addition of glucose to a glucosyltransferasereaction, in an amount equivalent to the amount of oligosaccharides usedin Example 3, is detrimental to the yield of insoluble alpha-1,3-glucanproduced by the glucosyltransferase reaction.

Glucan synthesis reactions were prepared as follows. Sucrose (75 g) wasweighed out and diluted to 0.75 L with deionized water in a 1-Lunbaffled jacketed flask that was connected to a LAUDA RK20recirculating chiller. Fermasure™ was then added (0.5 mL/L reaction),and the pH was adjusted to 5.5 using 5 wt % aqueous sodium hydroxide or5 wt % aqueous sulfuric acid. In comparative Example 4A (Table 8), thetrace amount of glucose came primarily from the sucrose component; noadditional glucose was added to the preparation. In Example 4.1 (Table8), glucose (18.8 g) was added to the preparation in addition to a traceamount of glucose present from the sucrose component. A polymerizationreaction was initiated in each preparation by adding 0.3 vol % of crudeGTFJ enzyme (SEQ ID NO:2) extract. Agitation to each reaction wasprovided using an overhead mechanical motor attached to a 4-blade PTFEblade, and the temperature was controlled at 25° C. After the reactionswere determined to be complete by HPLC, insoluble polymer product ofeach reaction was isolated by filtration. The polymer product was thenwashed with water (1.5 L), then washed with acetone (0.5 L), and thendried under a vacuum oven. The mass of the dry alpha-1,3-glucan productwas recorded.

Results for each reaction are shown in Table 8, demonstrating that theyield of insoluble alpha-1,3-glucan polymer obtained in aglucosyltransferase reaction is reduced when glucose is added to thereaction.

TABLE 8 Profiles of Glucosyltransferase Reactions Containing VariousAmounts of Glucose Example 4A 4.1 Initial sucrose (g/L) 97 96.32 Initialglucose (g/L) 2.5 27.8 % sucrose reacted 96.3 92.4 Yield polymer (g/L)17.4 13.6 Yield polymer (g/g sucrose reacted) 0.19 0.15

Thus, addition of glucose to a glucosyltransferase reaction isdetrimental to the yield of insoluble alpha-1,3-glucan produced by theglucosyltransferase reaction. It is noteworthy that the amount ofglucose in the reaction of Example 4.1 was equivalent to the amount ofoligosaccharides added to certain reactions in Example 3. The negativeresult in Example 4.1 thus indicates that it is oligomeric nature of theoligosaccharides used in Example 3 that is required for the observedglucan polymer yield-enhancing effect (i.e., the monomeric component,glucose, of the oligosaccharides most likely needs to be oligomerized inorder to enhance glucan product yield in a reaction).

Example 5 Insoluble Alpha-1,3-Glucan Yields in Reactions ContainingAdded Oligosaccharides and Different GTF Enzymes

This example discloses that the glucan polymer yield-enhancing effect ofadding purified oligosaccharides (from Example 2) to aglucosyltransferase reaction applies generally to reactions containingenzymes besides GTFJ that produce insoluble alpha-1,3-glucan.

The different types of GTF enzymes used in this example were GTF 0874(SEQ ID NO:4), GTF 1724-T1 (SEQ ID NO:7) and GTFJ-T1 (SEQ ID NO:8). Eachof these glucosyltransferases can synthesize, or is expected to be ableto synthesize, insoluble alpha-1,3-glucan polymer with about 100%alpha-1,3 glycosidic linkages (e.g., refer to U.S. Patent Appl. Nos.2014/0087431 and 2016/0002693, which are incorporated herein byreference).

Glucan synthesis reactions were prepared as follows. Sucrose (10 g),0.27 g dihydrogen potassium phosphate (KH₂PO₄), and 94 mL water wereadded to a 125-mL clean glass bottle equipped with a polypropylene cap.No oligosaccharides were added to the preparations in comparativeExamples 5A, 5B, and 5C (Table 9). In Examples 5.1, 5.2, and 5.3,certain amounts of the oligosaccharide solution prepared in Example 2(Table 5) were added to each respective preparation; the amount of wateradded to each respective preparation was reduced by an equivalentvolume. Each of the preparations contained a trace amount of glucosethat came primarily from the sucrose component; no additional glucosewas added to any of the preparations. Each preparation was agitated inan incubator shaker (temperature-controlled to ° C.) until a solutionformed, at which point the pH was adjusted to 5.5 using 5 wt % aqueoussodium hydroxide or 5 wt % aqueous sulfuric acid. A sample of eachpreparation was taken for analysis by HPLC, after which 0.3 vol % of acrude GTF enzyme extract prepared as described in the General Methodssection was added to each preparation to initiate a polymerizationreaction. Samples of each reaction were periodically taken and analyzedby HPLC as the reactions progressed. Once each reaction was deemedcomplete, the insoluble polymer product was isolated from the reactionby filtration, washed with 200 mL water, washed with 100 mL acetone, andthen dried.

Results for each reaction are shown in Table 9, which demonstrate thatthe yield of insoluble alpha-1,3-glucan polymer product is increasedwhen oligosaccharides were added to the reaction. This yield enhancementoccurred in reactions employing different types of glucosyltransferaseenzymes. It is notable that the approximate respective catalytic domainsof each of GTF 1724-T1 and GTF 0874 share roughly only 50% amino acidsequence identity with the approximate catalytic domain of GTFJ (referto U.S. Patent Appl. No. 2017/0002335, which is incorporated herein byreference). Despite this significant difference in sequence identity,each enzyme exhibited the insoluble alpha-1,3-glucan product yieldincrease.

TABLE 9 Profiles of Reactions Containing Different Types of GTF Enzymes,and Lacking or Containing Added Oligosaccharides Example 5A 5.1 5B 5.25C^(a) 5.3 Enzyme GTFJ-T1 GTF 1724-T1 GTF 0874 (SEQ ID (SEQ ID (SEQ IDNO: 8) NO: 7) NO: 4) Initial sucrose 141.0 150.2 149.5 150.2 142.6 150.2(g/L) Initial 0 5.7 0 5.7 0 5.7 oligosaccharides, DP2+ (g/L) Initialglucose, 1.8 0.8 0.6 0.8 4.1 0.8 (g/L) % sucrose reacted 93.7 96.4 99.599.4 90.0 97.6 Yield polymer 21.1 30.5 11.5 19.9 9.0 18.9 (g/L) Yieldpolymer 0.16 0.21 0.08 0.13 0.07 0.13 (g/g sucrose reacted)^(a)Comparative Example 5C was run at pH 7.0 instead of pH 5.5.

Thus, adding oligosaccharide by-products of an alpha-1,3-glucansynthesis reaction to a new alpha-1,3-glucan synthesis reaction canincrease the yield of alpha-1,3-glucan produced by the new reaction.This yield increase occurs in reactions employing various types ofglucosyltransferase enzymes.

Example 6 Insoluble Alpha-1,3-Glucan Yields in Reactions ContainingOther Types of Added Oligosaccharides

This example discloses that oligosaccharides likely must containalpha-1,3 glucosidic linkages to enable the shift in selectivity of aglucosyltransferase reaction towards insoluble alpha-1,3-glucan polymer.Oligosaccharides different from those produced in Example 2 (Table 5)were added to glucosyltransferase reactions to determine whether theycan affect alpha-1,3-glucan yield.

Maltodextrin (5.5, 15, or 18 dextrose equivalents; Sigma-Aldrich), whichhas 100% alpha-1,4 linkages and typically contains mostlyoligosaccharides (˜DP2-DP20), was used in alpha-1,3-glucanpolymerization reactions without further purification. Dextran (DextranT-10, average molecular weight of 10000 Daltons, Sigma Aldrich), whichis a polysaccharide containing >95% alpha-1,6 linkages, and hydrolyzeddextran, were also used in polymerization reactions. Hydrolyzed dextranwas prepared by heating a solution containing 15 g Dextran T-10 in 141mL water to 90° C. at pH 1.0. The distribution of oligosaccharides inthe hydrolyzed dextran preparation appears in Table 10.

TABLE 10 Composition of Hydrolyzed Dextran Preparation Glucose DP2 DP3DP4 DP5 DP6 DP7 DP8-10 DP10+ Total g/L 3.8 13.2 14.3 11.7 10.2 16.6 7.726.2 10.2 113.9 wt %^(a) 3.4 11.6 12.5 10.3  8.9 14.6 6.8 23.0  8.9100.0 ^(a)Weight percentage is with respect to the measured saccharidecomponents.

GTFJ reactions were conducted following the protocol described inExample 3, except that maltodextrin, dextran, or hydrolyzed dextran wasused instead of the oligosaccharides produced in Example 2 (Table 5).Table 11 provides the results of these reactions. Yields of insolublealpha-1,3-glucan polymer in reactions using hydrolyzed dextran (Examples6.1 and 6.2) and maltodextrins of various dextrose equivalents (Examples6.3-6.5) were not affected or only marginally affected by the additionof these different types of oligosaccharides (Table 11, compare Examples6.1-6.5 with Example 6A).

TABLE 11 Profiles of Glucosyltransferase Reactions Containing DifferentTypes of Added Oligosaccharides or Polysaccharides Example 6A 6.1 6.26.3 6.4 6.5 6.6 6.7 Oligosaccharide or — Hydrolyzed Maltodextrin,Maltodextrin, Maltodextrin, Dextran polysaccharide dextran DE^(b) 18DE^(b) 5.5 DE^(b) 15 T-10 tested Initial sucrose 97.2 98.6 102.1 100.4100.4 102.5 95.7 98.5 (g/L) Initial 0.0 1.6 2.3 6.5 6.5 6.2 6.2 27.9oligosaccharides (DP2+) or polysaccharides, (g/L) Initial glucose, 1.32.3 2.8 0.6 0.6 0.6 2.8 2.6 (g/L)^(a) % sucrose reacted 94.3 95.7 85.396.6 96.4 86.3 95.8 91.7 Yield polymer 15.4 15.9 14.2 17.5 17.4 10.821.8 23.7 (g/L) Yield polymer (g/g 0.17 0.17 0.16 0.18 0.18 0.12 0.240.26 sucrose reacted) ^(a)Trace glucose that was present in the sucrosecomponent of each reaction. ^(b)DE, dextrose equivalent.

The results in Table 11 indicate that oligosaccharides containingpredominantly alpha-1,6 linkages (hydrolyzed dextran, Examples 6.1 and6.2) or alpha-1,4 linkages (maltodextrin, Examples 6.3-6.5), when addedto glucosyltransferase reactions, do not significantly enhance the yieldof insoluble alpha-1,3-glucan polymer. Also, it appears that in orderfor an alpha-1,6-linked saccharide molecule to increase glucan productyield, such a saccharide molecule must be in the form of a largerpolysaccharide (10000 Daltons), since Dextran T-10 (Examples 6.6-6.7)increased insoluble alpha-1,3-glucan product yield, whereas itsoligosaccharide counterparts (Examples 6.1-6.2) did not.

Tables 6, 7 (Example 3), and 9 (Example 5) on the other hand indicatethat oligosaccharides comprising alpha-1,3 and alpha-1,6 linkages areable to significantly increase insoluble alpha-1,3-glucan yield inglucosyltransferase reactions. Based on these data, and thatoligosaccharides with only alpha-1,6 linkages did not significantlyaffect alpha-1,3-glucan product yield (Table 11), it appears that thealpha-1,3 linkage component of the oligosaccharides of Table 5 arerequired for enhancing insoluble alpha-1,3-glucan product yield.

Thus, oligosaccharides likely must contain at least some fraction ofalpha-1,3 glycosidic linkages to enhance insoluble alpha-1,3-glucanyield in a glucosyltransferase reaction.

Example 7 Isolation and Recycle of Oligosaccharides in Alpha-1,3-GlucanSynthesis Reactions

This example discloses that oligosaccharides generated from a glucanpolymerization reaction can be used to obtain consistent glucan productyield increases over multiple cycles of running polymerizationreactions.

A first glucan synthesis reaction was prepared as follows. Sucrose (75g) was weighed out and diluted to 0.75 L with deionized water in a 1-Lunbaffled jacketed flask that was connected to a LAUDA RK20recirculating chiller. Fermasure™ was then added (0.5 mL/L reaction),and the pH was adjusted to 5.5 using 5 wt % aqueous sodium hydroxide or5 wt % aqueous sulfuric acid. Purified oligosaccharides obtained from aglucan polymerization reaction (Table 5, Example 2) were added to thepreparation to a total concentration of DP2+ of ˜5 g/L. Agitation to thepreparation was provided using an overhead mechanical motor attached toa four-blade PTFE blade, and the temperature was controlled at 25° C. Apolymerization reaction was initiated by adding 0.3 vol % of crude GTFJenzyme (SEQ ID NO:2) extract. After the reaction was determined to becomplete by HPLC, insoluble polymer product was isolated by filtration.The polymer product was then washed with water (1.5 L), then washed withacetone (0.5 L), and then dried under a vacuum oven. The mass of dryalpha-1,3-glucan product was recorded.

The filtrate from the reaction was concentrated to ˜30 wt % dry solidsusing a rotary evaporator. A 25-mL fraction of this filtrate waspurified by column-chromatography using an ÄKTA EXPLORER system (GeneralElectric, Fairfield, Conn.). The run conditions of the chromatographicpurification are summarized in Table 12.

TABLE 12 Chromatographic Purification Run Conditions Resin Type BioRadBIO-GEL P-2 Gel Particle size (micron) 45-90 Bed length (cm) 100 Columndiameter (m) 0.026 Feed size (mL) 25 Approximate feed dry solids (g/100g) 30 Column temp (° C.) 50 Flow rate (mL/min) 50The fractions isolated from the chromatography were collected in 10-mLportions and analyzed by HPLC. Fractions containing oligosaccharideswere combined and concentrated by rotary evaporation at 40° C.

These purified oligosaccharides were then used as the oligosaccharidesource in a new glucan synthesis reaction (Example 7.1, Table 13)following the protocol of the first reaction (above); about 5 g/L ofoligosaccharides were provided to the reaction. After this reaction wascomplete, oligosaccharides (DP2+) were purified therefrom by the aboveprotocol and employed in a subsequent reaction. This cycle of runningglucan polymerization reactions comprising oligosaccharides (DP2+)purified from the immediate previous reaction was repeated an additionalfour times: oligosaccharides from the reaction of Example 7.1 were addedto the reaction of Example 7.2, oligosaccharides from the reaction ofExample 7.2 were added to the reaction of Example 7.3, oligosaccharidesfrom the reaction of Example 7.3 were added to the reaction of Example7.4, oligosaccharides from the reaction of Example 7.4 were added to thereaction of Example 7.5. Data from these experiments are summarized inTable 13, showing improved alpha-1,3-glucan yields over comparativeExample 7A, which did not have any oligosaccharides added to thereaction.

TABLE 13 Profiles of Glucosyltransferase Reactions UsingOligosaccharides Recycled from Previous Reactions Example 7A 7.1 7.2 7.37.4 7.5 Initial sucrose 141.5 152.8 152.0 152.4 154.0 150.4 (g/L)Initial 0.0 5.4 6.7 8.7 6.7 7.0 oligosaccharides, DP2+ (g/L) Initialglucose, 0.0 0.0 0.9 1.0 0.0 1.1 (g/L) % sucrose reacted 99.2 99.1 89.196.8 91.0 92.5 Yield polymer 23.4 27.6 29.2 29.1 26.8 33.0 (g/L) Yieldpolymer 0.17 0.18 0.22 0.20 0.19 0.24 (g/g sucrose reacted)Thus, based on the results shown in Table 13, oligosaccharides (DP2+)generated from a glucan polymerization reaction can be used to obtainfairly consistent glucan product yield increases over multiple cycles ofrunning polymerization reactions.

Example 8 Glucan Polymerization Reactions Using an Improved GTF Enzyme

This example discloses the oligosaccharide composition of filtrateproduced in an alpha-1,3-glucan synthesis reaction catalyzed by animproved glucosyltransferase.

The amino acid sequence of an S. salivarius glucosyltransferase enzymethat produces alpha-1,3-glucan with about 100% alpha-1,3 linkages wasmodified in its catalytic domain such that the enzyme could produce moreproducts (alpha-1,3-glucan and fructose), and less by-products (e.g.,glucose, oligosaccharides such as leucrose and DP2-7gluco-oligosaccharides), from sucrose substrate, as compared to theenzyme's unmodified counterpart (refer to U.S. Pat. Appl. Publ. No.2018/0072998, which is incorporated herein by reference).

An alpha-1,3-glucan synthesis reaction using the improvedglucosyltransferase was run in a 5000-gal stainless steel vesselcomprising 94 g/L white crystalline sucrose dissolved in water. The pHof the reaction was maintained using 10 mM potassium phosphate as abuffer and adjusted to 5.5 using 2 N H₂SO₄. An antimicrobial, FermaSure®XL, was added at 100 ppmv to prevent contamination during the reaction.The reactor contained three pitched blade impellers set to 33 rpm andwas controlled at 23° C. using cooled water flowing into the jacket ofthe reactor. The reaction was initiated by adding 30 pounds of theimproved glucosyltransferase enzyme, and deemed complete after 14 hoursat which time the sucrose concentration reached less than 2 g/L. At theend of the reaction, the glucosyltransferase enzyme was deactivated byheating the reaction contents to 65° C. for 30 minutes using an externalheat exchanger.

The insoluble alpha-1,3-glucan polymer (i.e., insoluble fraction)produced in the reaction was separated from the soluble fraction using afilter press, thereby providing a filtrate. Table 14 provides thecarbohydrate content (dwb) of the filtrate.

TABLE 14 Carbohydrate Composition of Filtrate (wt % - Dry Weight Basis)Fructose Glucose Leucrose DP2^(a) DP3+ Total 68.8 7.6 13.1 3.5 7.0 100^(a)Includes, in addition to DP2 gluco-oligosaccharides, at leastsucrose.

Chromatographic separation employing a strong acid cation-exchange resinwas used to isolate the oligosaccharide fraction of the filtrate. Thephysical parameters of the column used for this separation are in Table15.

TABLE 15 Physical Parameters of the Column Used for ChromatographicSeparation FINEX CS11GC, Resin Type #296 Ion form Na⁺ Crosslinking, %divinyl 5.5% benzene Particle size (mm) 0.35 Bed length (m) 5.2 Columndiameter (m) 0.225The filtrate was modified accordingly to 30 g dry solids/100 g solutionusing ion-exchanged tap water. Prior to addition of this modifiedfiltrate to the column resin, the resin was washed with six bed volumes(BV) of sodium chloride solution (three BV at 10 wt % sodium chloridefollowed by three BV at 5 wt % sodium chloride) to convert the resin tothe sodium form. The modified filtrate (15 L) was then fed to thecolumn, after which the column was eluted using ion-exchanged water at aflow rate of 30 L/h and at a column temperature of 70° C.

An oligosaccharide solution eluted between 140 and 185 minutes and wasrecovered. The oligosaccharide fraction thus prepared was analyzed byHPLC to determine its product distribution. Briefly, the composition ofthe oligosaccharide fraction was measured using an Agilent 1260 HPLCequipped with a refractive index detector. Separation of theoligosaccharides was realized using a BioRad AMINEX HPX-42A column usingwater as an eluent at 85° C. and a flow rate of 0.6 mL/min. Thecompositional profile of the oligosaccharides is provided in Table 16.

TABLE 16 Composition of Oligosaccharides Recovered by Fractionation (wt% - Dry Weight Basis) DP2 DP3 DP4 DP5 DP6 DP7+ 11 23 28 21 12 5

The oligosaccharide fraction described in Table 16 was subjected topartially methylated alditol acetate (PMAA) analysis (followingmethodology in Pettolino et al., Nature Protocols 7:1590-1607) andanalyzed by GC-MS. Briefly, the sample was treated with DMSO anion andiodomethane to methylate hydroxyl groups, and then hydrolyzed withtrifluoroacetic acid. The hydroxyl groups resulting from the brokenglycosidic linkages were then acetylated with acetic anhydride, and theresulting glucitols were analyzed by GC/MS. The oligosaccharides werefound to have the distribution described in Table 17 (all linkagestherein believed to be alpha). The dominant linkage was alpha-1,3. Noterminal fructose was detected in this oligosaccharide fraction.

TABLE 17 Linkage Distribution of Oligosaccharides Linkage Linkage % 1→387.5 1→6 7.3  1→3, 6 2.8 1→4 1.0  1→2, 3 0.7 1→2 0.6  1→3, 4 0.3

Thus, DP2+ oligosaccharides present in the filtrate from a glucansynthesis reaction employing an improved glucosyltransferase werecharacterized. Such oligosaccharides, or a filtrate comprising them, forexample, can be used as a source of added oligosaccharides forperforming a glucosyltransferase reaction as presently disclosed.

Example 9 Effect of Glucose, Leucrose, Fructose, orGluco-Oligosaccharide Additives on Alpha-1,3-Glucan Synthesis Reactions

This example discloses comparing the individual effects of varioussugars or oligosaccharides on enzymatic reactions for synthesizingalpha-1,3-glucan. Consistent with the above data (e.g., Examples 3, 5,7), this example shows that the addition of certain oligosaccharides toan alpha-1,3-glucan synthesis reaction can increase product yield.Further, the alpha-1,3-glucan product of this higher yield reaction hadsignificantly reduced intrinsic viscosity.

A 100 g/L sucrose/10 mM KH₂PO₄ solution (500 mL, pH-adjusted to 5.5 withsodium hydroxide or sulfuric acid) was added to individual 500-mL resinkettles serviced with overhead agitation. The following material wasthen added: glucose to 10 g/L, leucrose to 10 g/L, purifiedgluco-oligosaccharides to 10 g/L (produced similarly as in Example 8),fructose to 5 g/L, fructose to 10 g/L, fructose to 15 g/L, or fructoseto 30 g/L. One kettle did not receive any additional material and wasset as the control. The temperature of each kettle was adjusted to 25°C. Each reaction was then initiated by adding an aliquot (610 μL) of theglucosyltransferase enzyme used in Example 8 and allowed to run forabout 16 hours at 25° C. with moderate agitation. A sample from eachreaction was then taken, centrifuged and liquid-analyzed by HPLC forsugar content. The insoluble alpha-1,3-glucan produced in each reactionwas then filtered, washed with about 1 L of water, and dried for severaldays in a vacuum oven at 45° C. The reaction filtrates were discarded.

Greater than 99% of sucrose was converted in each reaction. Table 18provides the alpha-1,3-glucan yield of each reaction on an HPLC basis(difference in glucosyl consumption) and dried solids weight basis. Boththese yield measurements were fairly consistent with each other.

TABLE 18 Effect of Glucosyltransferase Reaction Additives onAlpha-1,3-Glucan Yield Reaction Alpha-1,3-Glucan Yield Additive HPLCbasis Dried Solids basis Control (no additive) 65% 63% Glucose (10 g/L)58% 59% Leucrose (10 g/L) 63% 63% Gluco-Oligosaccharides (10 g/L) 83%79% Fructose (5 g/L) 64% 60% Fructose (10 g/L) 62% 58% Fructose (15 g/L)60% 57% Fructose (30 g/L) 53% 52%Table 18 shows that alpha-1,3-glucan yield is reduced with the additionof glucose (consistent with Example 4) or increasing amounts offructose, the latter of which served to increase the yield of leucroseby-product (data not shown). While the addition of leucrose did not havean effect, the addition of gluco-oligosaccharides purified from aseparate glucosyltransferase reaction increased the yield ofalpha-1,3-glucan.

The molecular weight (DPw) and intrinsic viscosity (η, provided in mL/g)(abbreviated as “IV”) of alpha-1,3-glucan produced in some of the abovereactions were measured and are shown in Table 19. IV measurements inthe present Examples were made according to U.S. Pat. Appl. Publ. No.2017/0002335, which is incorporated herein by reference.

TABLE 19 Effect of Glucosyltransferase Reaction Additives onAlpha-1,3-Glucan Product IV and DPw IV Reaction Additive (mL/g) DPwControl-1 (no additive)^(a) — 780 Control-2 (no additive)^(b) 162 747Glucose (10 g/L) 157 713 Leucrose (10 g/L) 187 704Gluco-Oligosaccharides (10 g/L) 129 630 Fructose (30 g/L) 161 628^(a)Same control reaction as described above (Table 18). IV was notmeasured for the alpha-1,3-glucan product of this reaction. ^(b)Aseparate control reaction that was run in a similar manner to theControl-1 reaction.Table 19 shows that the IV of alpha-1,3-glucan is significantly reducedwith the addition of gluco-oligosaccharides purified from a separateglucosyltransferase reaction. Though the DPw of this alpha-1,3-glucanalso decreased, it is not believed this change accounts for the decreasein IV, as other additives also reduced DPw, but did not significantlyreduce IV. This is quite apparent with the fructose addition (30 g/L),for example, which effected virtually the same reduction inalpha-1,3-glucan DPw, but had no noticeable effect on IV.

Thus, adding gluco-oligosaccharide by-products of an alpha-1,3-glucansynthesis reaction to a new alpha-1,3-glucan synthesis reaction can both(i) increase the yield, and (ii) decrease the IV, of alpha-1,3-glucanproduced by the new reaction.

Example 10 Comparison of Alpha-1,3-Glucan Produced inGlucosyltransferase Reactions Lacking or Containing AddedGluco-Oligosaccharides

This example discloses that adding gluco-oligosaccharides generated froman alpha-1,3-glucan polymerization reaction can be used to obtain analpha-1,3-glucan product with lower aqueous slurry viscosity and lowerdissolved polymer solution viscosity than alpha-1,3-glucan produced witha reaction that otherwise does not have the addedgluco-oligosaccharides.

Each of the reactions prepared in this example employed theglucosyltransferase used in Example 8.

A first alpha-1,3-glucan reaction without the addition ofgluco-oligosaccharides was prepared in a 4-L jacketed glass reactor withoverhead stirring and an external chiller/heater to maintain a constanttemperature. The reaction media was prepared by adding 299 g of sucroseto 2412 g of water, after which 3.54 g of potassium phosphate and 130 μLof Fermasure® were added; the solution pH was then adjusted to 5.5 usingsodium hydroxide or sulfuric acid. The reactor was maintained at aconstant temperature of 20° C. with constant stirring with three 45°pitched blade impellers stirring at 250 rpm. The reaction was initiatedby addition of 0.1 vol % glucosyltransferase enzyme solution to thestirred solution. The reaction was completed when the sucrose was below5 g/L, after which the entire reactor was heated above 65° C. for aminimum of 1 hour followed by cooling down to room temperature.

The alpha-1,3-glucan produced in the first reaction was filtered in avacuum Buchner funnel with filter paper, and the filtrate (whichcontains gluco-oligosaccharides) was collected to be used in thesubsequent reaction. The alpha-1,3-glucan cake was then washed andfiltered with more than 8 L of water to separate the sugars from thealpha-1,3-glucan to provide a cake with greater than 10 wt % solids(percent solids was measured accordingly).

A second reaction was prepared in the same reactor vessel by adding 299g of sucrose to 780 g of filtrate from the first reaction and 1632 g ofwater. Since the filtrate contained 1.03 g of potassium phosphate, 2.51g of potassium phosphate was added to the second reaction. The solutionwas mixed and temperature controlled to 20° C. followed by the additionof 130 μL of Fermasure® and 0.1 vol % glucosyltransferase enzymesolution. The heating and filtration steps from the first reaction wererepeated for the second reaction.

A third reaction was set up that was a repeat of the second reaction,but using filtrate collected from the second reaction. Table 20summarizes the components of each of the first-third reactions(Reactions 1-3, respectively).

TABLE 20 Saccharide Components of Reactions 1-3 Component Reaction 1Reaction 2 Reaction 3 Initial Sucrose (g/L) 114 115 116 Initial Leucrose(g/L) 0.0 4.9 9.0 Initial Glucose(g/L) 0.5 2.0 1.4 Initial Fructose(g/L)0.3 16.0 19.6 Initial Gluco-Oligosaccharides(g/L) 0.0 3.5 3.9

The aqueous slurry viscosity of the alpha-1,3-glucan products of each ofthe first, second and third reactions was measured by first addingglucan cake with enough water to make a 4 wt % aqueous mixture, and thenhomogenizing the mixture. The viscosity of each mixture was thenmeasured on a rheometer at 20° C., ramping the shear rate from 7 s⁻¹ to200 s⁻¹ and measuring viscosity in centipoise (cP). The measurements areshown in FIG. 2 , which shows a decrease in aqueous slurry viscosity ofthe alpha-1,3-glucan products as successively made in the first throughthird reactions.

The molecular weight (DPw) and intrinsic viscosity (IV) of each of thealpha-1,3-glucan products of the first-third reactions were measured(Table 21, Reactions 1-3, respectively) following their dissolution to aconcentration of 10 mg/mL in DMAc/0.5% LiCl.

TABLE 21 Molecular Weight and IV of the Alpha- 1,3-Glucan Products ofReactions 1-3 Reaction 1 Reaction 2 Reaction 3 DPw 773 753 736 IV (mL/g)292 262 248

Thus, consistent with the results of Example 9 above, addinggluco-oligosaccharide by-products of an alpha-1,3-glucan synthesisreaction to a new alpha-1,3-glucan synthesis reaction can decrease theviscosity of alpha-1,3-glucan (as measured in both aqueous slurry anddissolved polymer formats) produced by the new reaction.

Example 11 Comparison of Alpha-1,3-Glucan Polymers Produced inGlucosyltransferase Reactions in Which Gluco-Oligosaccharides are Addedin Batch or Fed-Batch Manner

This example discloses that adding gluco-oligosaccharides generated froman alpha-1,3-glucan polymerization reaction can be used in furtherreactions in either batch or fed-batch mode. In particular, this examplediscloses that adding gluco-oligosaccharides over the course analpha-1,3-glucan polymerization reaction (fed-batch addition) reducesthe viscosity of the glucan polymer produced over the reaction time.However, the final alpha-1,3-glucan polymer produced at the end of thereaction has a higher viscosity than alpha-1,3-glucan polymer producedin a reaction where all the added gluco-oligosaccharides are provided ina batch at the beginning of the reaction (batch addition). The higherfinal intrinsic viscosity (IV) of the polymer product of the fed-batchmode reaction is likely due to the reaction's lower initialgluco-oligosaccharide concentration compared to that of the batchreaction.

Each of the reactions prepared in this example employed theglucosyltransferase used in Example 8. The gluco-oligosaccharides usedin these reactions were provided in the form of a glucosyltransferasereaction filtrate as prepared in Example 10, for example.

A fed-batch reaction was prepared in a 4-L jacketed glass reactor withoverhead stirring and an external chiller/heater to maintain a constanttemperature. The reaction media was prepared by adding 260 g of sucroseto 1656 g of water, after which 2.51 g of potassium phosphate and 130 μLof Fermasure® were added; the solution pH was then adjusted to 5.5 usingsodium hydroxide or sulfuric acid. The reactor was maintained at aconstant temperature of 23° C. with constant stirring with three 45°pitched blade impellers at 200 rpm. The reaction was initiated byaddition of 0.1 vol % glucosyltransferase enzyme solution to the stirredsolution. Gluco-oligosaccharides were added at a rate of 78 mL/hr afterthe start of the reaction. Samples were removed from the reactor everyhour for the first six hours; alpha-1,3-glucan product in each samplewas separated from the liquid by filtration and then washed three timeswith water. The reaction was completed when the sucrose was below 5 g/L(˜22 hours), after which the entire reactor was heated above 65° C. fora minimum of 1 hour followed by cooling to room temperature.

A batch reaction was prepared in the same reactor vessel with 260 g ofsucrose mixed with 1656 g of water and 780 g of liquid containinggluco-oligosaccharides. The solution was mixed and temperaturecontrolled to 20° C. followed by the addition of 130 μL of Fermasure®and 0.1 vol % glucosyltransferase enzyme solution. Samples were obtainedand processed, and the reaction was terminated, in the same manner asdone with the fed-batch reaction.

Table 22Error! Reference source not found. shows the change ingluco-oligosaccharide concentration during the fed-batch and batchreactions, and confirms that the initial gluco-oligosaccharideconcentration was higher initially in the batch reaction compared to thefed-batch reaction.

TABLE 22 Gluco-Oligosaccharide Concentrations during the Fed-Batch andBatch Reactions Gluco-Oligosaccharide Reaction Concentration (g/L) TimeFed-Batch Batch (hr) Reaction Reaction 0 0 4 1 2 5 2 4 5 3 5 6 4 7 7 5 88 6 9 9

The molecular weight (MW) and intrinsic viscosity (IV) of the respectivealpha-1,3-glucan products of the fed-batch and batch reactions weremeasured (Tables 23 and 24) following their dissolution to aconcentration of 10 mg/mL in DMAc/0.5% LiCl.

TABLE 23 Viscosity of the Alpha-1,3-Glucan Products of Fed-Batch andBatch Reactions Reaction IV Reaction Time (hr) (mL/g) Fed-Batch 1 331Fed-Batch 2 284 Fed-Batch 3 256 Fed-Batch 4 212 Fed-Batch 5 222Fed-Batch 6 225 Fed-Batch 22 205 Batch 22 174Table 23 shows that there was a decrease in alpha-1,3-glucan polymerviscosity as a function of time for the fed-batch reaction. However, thefed-batch final alpha-1,3-glucan viscosity was higher than the batchfinal alpha-1,3-glucan viscosity (both measured at 22-hour timepoints,Table 23).

TABLE 24 Molecular Weight of the Alpha-1,3-Glucan Products of Fed-Batchand Batch Reactions Reaction DPw Time (hr) Fed-Batch Batch 1 1130 876 21021 849 3 955 816 4 905 783 5 848 771 6 831 746 22 753 682

Thus, consistent with the results of Examples 9-10 above, addinggluco-oligosaccharide by-products of an alpha-1,3-glucan synthesisreaction in either batch or fed-batch mode to a new alpha-1,3-glucansynthesis reaction can decrease the viscosity of alpha-1,3-glucanproduced by the new reaction. It is noteworthy, though, that suchaddition in batch mode has a greater effect on reducing polymerviscosity.

Example 12 Comparison of Alpha-1,3-Glucan Polymers Produced inGlucosyltransferase Reactions with Added Gluco-Oligosaccharides atVarying Temperatures

This example discloses that adding gluco-oligosaccharides generated froman alpha-1,3-glucan polymerization reaction to other alpha-1,3-glucanpolymerization reactions at varying temperatures reduces the viscosityof the glucan polymer products of the latter reactions. This change inviscosity was significantly higher at lower reaction temperatures.

Each of the reactions prepared in this example employed theglucosyltransferase used in Example 8. The gluco-oligosaccharides usedin these reactions were provided in the form of a glucosyltransferasereaction filtrate as prepared in Example 10, for example.

Reactions were run in 500-mL jacketed glass reactor with overheadstirring and an external chiller/heater to maintain a constanttemperature. The reaction media was prepared by adding 50 g of sucroseto 469 g of water, after which 0.68 g of potassium phosphate and 25 μLof Fermasure® were added; the solution pH was then adjusted to 5.5 usingsodium hydroxide or sulfuric acid. The reactors were maintained at aconstant temperature with constant stirring with three 45° pitched bladeimpellers at 200 rpm. Each reaction was initiated by addition of 0.1 vol% glucosyltransferase enzyme solution to the stirred solution. Eachreaction was completed when the sucrose was below 5 g/L, after which theentire reactor was heated above 65° C. for a minimum of 1 hour followedby cooling to room temperature.

The reactions (1-9) were run with three temperatures and threeconcentrations of gluco-oligosaccharides. The gluco-oligosaccharideconcentration was changed by addition of appropriate amounts of filtratefrom a previous alpha-1,3-glucan polymerization. The liquid added toeach reaction was an appropriate mixture of water and filtrate. Table 25shows the reaction temperatures and initial gluco-oligosaccharideconcentrations of reactions 1-9. Following completion of all thereactions, the alpha-1,3-glucan products were filtered and washed withmore than 1 L of water to prepare glucan wet cakes with greater than 10wt % solids. Each cake was dissolved to a concentration of 10 mg/mL inDMAc/0.5% LiCl, after which the molecular weight and intrinsic viscosityof the glucan polymer products were measured (Table 25).

TABLE 25 Molecular Weight and Viscosity of Alpha-1,3-Glucan Produced inReactions with Varying Temperature and Initial Gluco-OligosaccharideConcentrations Initial Reaction Gluco-Oligosaccharide TemperatureConcentration IV Reaction (° C.) (g/L) DPw (mL/g) 1 10 0.65 985 325 2 103.03 923 264 3 10 5.08 795 228 4 20 0.91 896 221 5 20 3.41 804 196 6 205.13 753 176 7 30 0.51 515 138 8 30 2.67 469 128 9 30 4.54 432 119

Table 25 shows that alpha-1,3-glucan product viscosity is lower inreactions (held at the same temperature) with higher initialgluco-oligosaccharide concentrations, which is consistent with aboveresults. It is evident that this viscosity change was more pronounced(percentage-wise) in reactions held at a lower temperature.

Example 13 Alpha-1,3-Glucan Polymer Produced in GlucosyltransferaseReactions with Delayed Addition of Gluco-Oligosaccharides

This example discloses that adding gluco-oligosaccharides generated froman alpha-1,3-glucan polymerization reaction to another alpha-1,3-glucanpolymerization reaction four hours after the latter reaction hascommenced (initiated by addition of glucosyltransferase enzyme) producesglucan polymer with similar viscosity compared to alpha-1,3-glucanpolymerization reactions with no added gluco-oligosaccharides.

The reaction prepared in this example employed the glucosyltransferaseused in Example 8. The gluco-oligosaccharides used in this reaction wereprovided in the form of a glucosyltransferase reaction filtrate asprepared in Example 10, for example.

A reaction was prepared in a 500-mL jacketed glass reactor with overheadstirring and an external chiller/heater to maintain a constanttemperature. The reaction media was prepared by adding 46 g of sucroseto 364 g of water, after which 0.44 g of potassium phosphate and 204 ofFermasure® were added; the solution pH was then adjusted to 5.5 usingsodium hydroxide or sulfuric acid. The reactor was maintained at aconstant temperature of 19° C. with constant stirring with three 45°pitched blade impellers at 150 rpm. The reaction was initiated by adding0.1 vol % glucosyltransferase enzyme solution to the stirred solution.Four hours after reaction initiation, liquid containing 11.5 g ofsucrose, 0.11 g potassium phosphate, and 100 g of liquid withgluco-oligosaccharides was added to the reaction. The reaction wascompleted when the sucrose was below 5 g/L, after which the entirereactor was heated above 65° C. for a minimum of 1 hour followed bycooling to room temperature.

Following completion of the reaction, the alpha-1,3-glucan product wasfiltered and washed with more than 1 L of water to prepare a glucan wetcake with greater than 10 wt % solids. The cake was dissolved to aconcentration of 10 mg/mL in DMAc/0.5% LiCl, after which the molecularweight and intrinsic viscosity of the glucan polymer product weremeasured (Table 26).

TABLE 26 Molecular Weight and Viscosity of Alpha-1,3-Glucan Produced ina Reaction with Delayed Gluco-Oligosaccharide Addition IV ReactionDescription DPw (mL/g) Delayed Gluco-Oligosaccharide 830 219 Addition(this Example) Modest Gluco-Oligosaccharide Addition 896 221 (Example12, Reaction 4) Gluco-Oligosaccharide Addition at 753 176 ReactionBeginning (Example 12, Reaction 6)

It is apparent from Table 26 that adding gluco-oligosaccharidesgenerated from an alpha-1,3-glucan polymerization reaction to anotheralpha-1,3-glucan polymerization reaction some time after the latterreaction has commenced produces glucan polymer with similar viscositycompared to alpha-1,3-glucan polymerization reactions with only a modestamount of added gluco-oligosaccharides.

What is claimed is:
 1. A method for producing insoluble alpha-1,3-glucancomprising: (a) providing filtrate or supernatant of aglucosyltransferase reaction that produces insoluble alpha-1,3-glucanhaving at least 50% alpha-1,3 glycosidic linkages; and (b) contacting atleast sucrose, said filtrate or supernatant, and a glucosyltransferaseenzyme that produces insoluble alpha-1,3-glucan having at least 50%alpha-1,3 glycosidic linkages, thereby producing said insolublealpha-1,3-glucan; wherein the glucosyltransferase of theglucosyltransferase reaction of step (a) comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:2, 4, 6, 7, 8, or9, and the glucosyltransferase of step (b) comprises an amino acidsequence that is at least 90% identical to SEQ ID NO:2, 4, 6, 7, 8, or9.
 2. The method of claim 1, wherein the glucosyltransferase reaction ofstep (a) produces insoluble alpha-1,3-glucan having at least 80%alpha-1,3 glycosidic linkages, and the glucosyltransferase of step (b)produces insoluble alpha-1,3-glucan having at least 80% alpha-1,3glycosidic linkages.
 3. The method of claim 2, wherein theglucosyltransferase reaction of step (a) produces insolublealpha-1,3-glucan having at least 90% alpha-1,3 glycosidic linkages, andthe glucosyltransferase of step (b) produces insoluble alpha-1,3-glucanhaving at least 90% alpha-1,3 glycosidic linkages.
 4. The method ofclaim 3, wherein the glucosyltransferase reaction of step (a) producesinsoluble alpha-1,3-glucan having at least 95% alpha-1,3 glycosidiclinkages, and the glucosyltransferase of step (b) produces insolublealpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages. 5.The method of claim 1, wherein the glucosyltransferase reaction of step(a) produces insoluble alpha-1,3-glucan having a weight-average degreeof polymerization (DPw) of at least 100, and the glucosyltransferase ofstep (b) produces insoluble alpha-1,3-glucan having a DPw of at least100.
 6. The method of claim 2, wherein the glucosyltransferase reactionof step (a) produces insoluble alpha-1,3-glucan having a weight-averagedegree of polymerization (DPw) of at least 100, and theglucosyltransferase of step (b) produces insoluble alpha-1,3-glucanhaving a DPw of at least
 100. 7. The method of claim 3, wherein theglucosyltransferase reaction of step (a) produces insolublealpha-1,3-glucan having a weight-average degree of polymerization (DPw)of at least 100, and the glucosyltransferase of step (b) producesinsoluble alpha-1,3-glucan having a DPw of at least
 100. 8. The methodof claim 4, wherein the glucosyltransferase reaction of step (a)produces insoluble alpha-1,3-glucan having a weight-average degree ofpolymerization (DPw) of at least 100, and the glucosyltransferase ofstep (b) produces insoluble alpha-1,3-glucan having a DPw of at least100.
 9. The method of claim 1, wherein the yield of the insolublealpha-1,3-glucan produced is increased compared to the yield ofinsoluble alpha-1,3-glucan that would be produced if step (b) lacked thefiltrate or supernatant.
 10. The method of claim 1, wherein theviscosity of the insoluble alpha-1,3-glucan produced is decreasedcompared to the viscosity of insoluble alpha-1,3-glucan that would beproduced if step (b) lacked the filtrate or supernatant, whereinviscosity is measured with alpha-1,3-glucan as mixed or dissolved in aliquid.
 11. The method of claim 1, wherein the glucosyltransferasereaction of step (a) is at least about 90% complete as measured by itspercent sucrose consumption.
 12. The method of claim 1, wherein steps(a) and (b) are repeated one or more times, and the filtrate orsupernatant in each repeated step (a) is provided from theglucosyltransferase reaction of a preceding step (b).
 13. The method ofclaim 1, wherein the glucosyltransferase of the glucosyltransferasereaction of step (a) comprises an amino acid sequence that is at least95% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and theglucosyltransferase of step (b) comprises an amino acid sequence that isat least 95% identical to SEQ ID NO:2, 4, 6, 7, 8, or
 9. 14. The methodof claim 13, wherein the glucosyltransferase of the glucosyltransferasereaction of step (a) comprises an amino acid sequence that is at least97% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and theglucosyltransferase of step (b) comprises an amino acid sequence that isat least 97% identical to SEQ ID NO:2, 4, 6, 7, 8, or
 9. 15. The methodof claim 14, wherein the glucosyltransferase of the glucosyltransferasereaction of step (a) comprises an amino acid sequence that is at least98% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and theglucosyltransferase of step (b) comprises an amino acid sequence that isat least 98% identical to SEQ ID NO:2, 4, 6, 7, 8, or
 9. 16. The methodof claim 1, wherein the method further comprises isolating the insolublealpha-1,3-glucan produced in step (b).
 17. The method of claim 1,wherein step (a) comprises providing said filtrate.
 18. The method ofclaim 1, wherein step (a) comprises providing said supernatant.
 19. Themethod of claim 12, wherein said filtrate in each repeated step (a) isprovided.